Method and apparatus for antibody production and purification

ABSTRACT

The subject invention pertains to methods and apparatus for the production and purification of cell products, such as immunoglobulins. One aspect of the invention is an integrated cell culture and purification apparatus for the growth and maintenance of cells and the harvest and purification of cell products, such as immunoglobulins. Thus, the apparatus integrates a cell culture function with a purification function. Other aspects of the invention pertain to an automated method for producing immunogenic compositions such as vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/516,868, filed Oct. 17, 2014, which is a continuation of U.S.application Ser. No. 14/122,327, filed Nov. 26, 2013, which is theNational Stage of International Application No. PCT/US2012/041949, filedJun. 11, 2012, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/495,832, filed Jun. 10, 2011, each of which is herebyincorporated by reference herein in its entirety, including any figures,tables, or drawings.

BACKGROUND OF THE INVENTION

The anticipated growth of personalized medicine will require newparadigms for the manufacture of therapies tailored to the needs ofindividual patients. The greatest challenge is expected to come in thearea of cell based therapies, especially when such therapies areautologous in nature. In such cases, each cell or cell based productwill need to be manufactured from scratch for each patient. Manualmethods for mammalian cell culture, by their nature, are prone totechnician error or inconsistency leading to differences betweensupposed identical cultures. This becomes especially evident as more andmore autologous cells are expanded for personalized therapies.Patient-specific cells, or cell products such as proteins, are subjectto variation, especially when scaled beyond levels that can be managedefficiently with manual methods.

With the increased use of proteins, such as antibodies, in clinicaldiagnostics and therapy, the need has arisen for more efficient, rapidand sterile purification methods. Conventional purification techniquestypically involve manually passing mixtures containing proteins througha suitable column to selectively adsorb the proteins from the mixture.The adsorbed proteins are then eluted from the column in purified form.

Manual methods for purifying proteins have their drawbacks. For example,these methods can be labor intensive, time consuming and typically usemultiple columns which are manually packed with resin and sterilizedprior to each purification run. The manual steps involved in thesemethods also include a high risk of contamination.

In addition to being labor intensive, the stringent requirements forsegregation of each patient's materials from that of every other patientwill mean that manufacturing facilities will be large and complex,containing a multitude of isolation suites each with its own equipment(incubators, tissue culture hoods, centrifuges) that can be used foronly one patient at a time. Because each patient's therapy is a new andunique product, patient specific manufacturing will also be laborintensive, requiring not just direct manufacturing personnel but alsodisproportionately increased manpower for quality assurance and qualitycontrol functions.

Moreover, conventional approaches and tools for manufacturing cells orcell based products typically involve numerous manual manipulations thatare subject to variations even when conducted by skilled technicians.When used at the scale needed to manufacture hundreds or thousands ofdifferent cells, cell lines and patient-specific cell based therapies,the variability, error or contamination rate may become unacceptable forcommercial processes.

Small quantities of cell-secreted product are produced in a number ofdifferent ways. T-flasks, roller bottles, stirred bottles or cell bagsare manual methods using incubators or warm-rooms to provideenvironments for cell growth and production. These methods are verylabor intensive, subject to mistakes and difficult for large-scaleproduction.

Another method for producing cell secreted products is by ascitesproduction, which requires injecting the peritoneum of a host animal(usually a mouse) with the cells that express the product, which arethereby parasitically grown and maintained. The animals are sacrificedand the peritoneal fluid with the product is collected. This method isalso very labor intensive, difficult for large scale production, andobjectionable because of the use of animals.

Another method for producing cell secreted products involves inoculatingand growing the cells in a small stirred tank or bioreactor or bag-typechamber. The tank provides the environmental and metabolic needs and thecell secretions are allowed to accumulate. This method is costly interms of facility support in order to accommodate a large number ofunique cells and produces product at low concentration.

Another method for the production of cell secreted products is to use abioreactor (hollow fiber, ceramic matrix, fluidizer bed, etc.) in lieuof the stirred tank. This can bring facilities costs down and increasesproduct concentration. The systems currently available are generalpurpose in nature and require considerable time from trained operatorsto setup, load, flush, inoculate, run, harvest, and unload. Each steptypically requires manual documentation, which is labor intensive andsubject to errors.

Cell culturing devices or cultureware for culturing cells in vitro areknown. As disclosed in U.S. Pat. No. 4,804,628, the entirety of which ishereby incorporated by reference, a hollow fiber culture device includesa plurality of hollow fiber membranes. Medium containing oxygen,nutrients, and other chemical stimuli is transported through the lumenof the hollow fiber membranes or capillaries and diffuses through thewalls thereof into an extracapillary (EC) space between the membranesand the shell of the cartridge containing the hollow fibers. The cellsthat are to be maintained collect in the extracapillary space. Metabolicwastes are removed from the bioreactor. The cells or cell products canbe harvested from the device.

Known EC reservoirs have typically been rigid. They are a pressurevessel and therefore require a sealed compartment with tubing portsadding to costs. A gas, typically air, is introduced through a sterilebarrier, generally a membrane filter, to control pressure in the vessel.Fluid level control has been limited to ultrasonic, conductive oroptical trip points, or by a load cell measuring the weight of thefluid. Reservoirs are expensive and difficult to manufacture. There islimited EC fluid level measurement accuracy-ultrasonic, conductive oroptical monitoring of fluid levels are commonly fouled by cell debris inthe reservoir. Alternatively, load cells are not a rugged design forreliable fluid level sensing.

Another problem with the prior systems is the inability to controllactate and sense pH in the system. One method takes samples of theculture medium and analyzes it using an off-line analyzer. The operatoradjusts the perfusion medium rate based on values obtained to maintainthe lactate concentration at the level desired. The operator mustattempt to predict future lactate levels when adjusting media feedrates. This is labor intensive, presents potential breech of sterility,and the level of lactate control accuracy is dependent on operatorskill.

Another method is to connect an automated sampler/analyzer toperiodically withdraw sample of the culture media, analyze it andprovide feedback for a media feed controller. This method requiresadditional equipment and increases the risk of sterility breech.

Yet another method is to use an invasive lactate sensor to directly readthe lactate level and provide feedback for a media feed controller. Inline lactate sensors need to be sterilizable, biocompatible, typicallyhave low reliability and need periodic maintenance.

These methodologies rely on costly, labor intensive off-line samplingand analysis or additional equipment to interface with the instrument orrequire the addition of a lactate probe and electronics to the culture.

Disposable cultureware generally cannot be autoclaved, so a pH sensor ishistorically sterilized separately and then added to the cultureware.However, adding the probe risks compromising the sterility of thecultureware. Probe addition is performed in a sterile environment(laminar flow hood) and increases the manpower needed.

The previous methodologies that utilize off-line sampling are subject tocontamination problems and depend on the skill of the operator inpredicting future lactate levels and influence of media dilution rate.Sampling equipment need interfacing to the culture fluidic circuit, aninterface for the feedback signal and periodic calibration of the probesused for sampling. The lactate probe requires interface with the fluidcircuit, a method for sterilization or a sterile barrier, interfaceelectronics to convert the probe signal to a useful feedback and amethod to calibrate in the fluid circuit.

Preparing conventional systems to start the cell culture is also verylabor intensive. The cultureware must be assembled and sterilized orprobes must be prepared, sterilized and aseptically inserted into thepre-sterilized portion of the cultureware. The cultureware assembly isthen loaded onto the instrument. A series of manual operations areneeded to check the integrity of the assembly, introduce fluid into thecultureware flow path, flush the toxic residuals (e.g., surfactants)from the cultureware, start the cultureware in a pre-inoculation mode,introduce factors into the flow path getting it ready for the cells,inoculating the cells into the bioreactor and starting the run (growthof the cell mass and eventual harvest of product).

Two methods are generally used for sterilization. One method places anelectrode in a holder, steam sterilizes the assembly (probe) and thenaseptically inserts the probe into the pre-sterilized cultureware. Thesecond method involves placing a non-sterile probe into a holder andthen using steam to sterilize the electrode in place, referred to assteam in place. Both methods are labor intensive, prone to failure andthe procedures need to be validated.

Other methods exist which are less common. Cold sterilants can be usedto sterilize the holder and electrode before aseptic insertion. Apermeable membrane can be used to isolate the non-sterile probe from thesterile fluid being sensed. A holder with the membrane is placed in thefluid path, either before sterilization or after if the holder andmembrane is sterilized separately, and then the sensor is placed againstor close to the membrane and the fluid on both sides of the membrane isassumed to be equilibrated.

Glass electrodes have not been included with the cultureware in the pastbecause it was unknown if the probes could survive EtO sterilization andbeing stored dry. Filled glass electrodes are normally stored hydratedin a liquid buffer.

Each unique cell or cell line must be cultured, with cell productsharvested and purified separately. In order to accommodate a largenumber of unique cells or cell lines, a considerable number ofinstruments would be needed. If application of the cells or products fortherapeutic purposes is contemplated, strict segregation of each cellproduction process would be required. Consequently, compactness of thedesign and the amount of ancillary support resources required willbecome an important facilities issue. Moreover, the systems currentlyavailable are general purpose in nature and require considerable timefrom trained operators to setup, load, flush, inoculate, run, harvestand unload. Each step usually requires manual documentation.

Moreover, production tracking mandates generation of a batch record foreach cell culture run. Historically, this is done with a paper-basedsystem and relies on the operator inputting the information. This islabor intensive and subject to errors.

Current purification techniques also involve cleaning and reuse ofcomponents. This requires Standard Operational Procedures (SOPs) to bewritten and the cleaning and reuse process to be validated. This is atime intensive activity.

Accordingly, there is a need for an apparatus and method whereby cellsand/or cell products can be cultured and purified in a fully automated,rapid and sterile manner.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention is an integrated cell culture andpurification apparatus for the growth and maintenance of cells and theharvest and purification of cell products such as cell secreted products(if required). Thus, the apparatus integrates a cell culture functionwith a purification function. The apparatus can be used for shortproduction runs (e.g., less than 45 days) of a large number of uniquecell lines, or longer production runs. The apparatus is designed suchthat it can be incorporated into a production facility, and to supportcentralized batch information gathering. Advantageously, the apparatus'sdesign minimizes operator time needed to setup and run the apparatus.Purification of cell-secreted product is exemplified herein; however, ifcell production is the product, a cell harvest unit can be used in placeof the purification unit.

The integrated apparatus of the invention has the capacity for a highdegree of automation. This reduces the amount of operator's time neededand reduces operator-induced errors. Cultureware modules (disposableportions of the cell culture unit and purification unit) are highlyintegrated and easy to load/unload. The control process is designed tocomplete sequences without the need for operator's intervention. Theapparatus is modularized into a cell culture unit and purification unit.The purification unit uses the resources of the cell culture module forcoordination and control. A single purification unit can supportmultiple cell culture units. The apparatus is designed to facilitate acentral electronic records system operated in a current goodmanufacturing practice (CGMP) facility. Instrument support is providedfor operator and consumables code identification (e.g., bar codeidentification, radio frequency identification (RFID) tagidentification, bokode, identification or quick response (QR) codeidentification).

The integrated apparatus of the invention includes two units: a cellculture unit and a purification unit. The two units are physicallyseparate but designed to be placed together (e.g., adjacent to oneanother). The cell culture unit and purification unit can transfer dataand coordinate activity with each other using methods known in the art,such as through an infrared communication port. The purification unitcan be placed next to the cell culture unit towards the end of theproduction period. A tubing line from each unit's cultureware connectstogether to provide a fluid path for the collected harvest fluid in thecell culture unit to be transferred to the purification unit. Theoperator initiates the purification sequence through a user interfacesuch as a touch screen interface on the cell culture unit. The endproduct of the system can be a purified and neutralized product in abuffered solution suitable for further processing.

The cell culture unit of the integrated apparatus includes twoindividual parts: a reusable instrumentation base device (also referredto herein as a “first” reusable instrumentation base device, todistinguish it from the reusable instrumentation base device of thepurification unit), and at least one disposable cell cultureware modulethat is used for a single production run and is disposable (alsoreferred to herein as a “first” disposable cell cultureware module, todistinguish it from the at least one disposable cultureware of thepurification unit). The instrumentation base device provides thehardware to support cell culture growth and production in a compactpackage, which is advantageous in a facility handling a large number ofunique cell lines, for example. A pump, such as an easy-load 4 channelperistaltic pump, moves fresh basal media into the cultureware, removesspent media, adds high molecular weight factor and removes productharvest. An integrated cool storage area maintains the factor andharvest at a low temperature (preferably, approximately 4° C.). Aheating mechanism maintains the cell environment to promote growth andproduction. The gas blending mechanism, in conjunction with thecultureware pH sensor controls the pH of the cell culture medium. Aplurality of automated tube valving drives (e.g., three automated tubevalving drives) are used to control the cultureware flowpathconfiguration to accomplish the fluidic functions necessary to initiateand carryout a successful run. Valves and sensors in the instrumentationbase device control the fluid cycling in the cultureware. Drive forfluid circulation is provided. An identification code reader, such as abarcode reader, is preferably included to facilitate operator and lottracing. A communication port preferably ties the instrumentation deviceto a facilities data management system (LIMS). Preferably, theinstrumentation device of the cell culture unit includes a userinterface, such as a flat panel display with touch screen, for userinteraction.

The one-time use cultureware is provided pre-sterilized, designed forrapid loading onto the instrumentation base device (“quick-load”). Theloading of the cultureware body makes connections to the instrumentationbase device. The pump cassette, which is physically attached to thetubing, allows the user to quickly load the pump segments. The designand layout minimizes loading errors. The cultureware enclosure providesan area that is heated to maintain cell fluid temperature. Reservoirs tomaintain fluid volumes and cycling are included in the cultureware.Sensors for fluid circulation rate and pH and thermal well for theinstrument's temperature sensor are included. The blended gas from theinstrumentation device is routed to the gas exchange cartridge thatprovides oxygen and adds or removes carbon dioxide to the circulatedfluid to support cell metabolism. The cultureware module also includes abioreactor (e.g., hollow fiber bioreactor or other bioreactor type),which provides the cell space and media component exchange. Disposablecontainers for harvest collection and flushing are provided. Theoperator attaches a media source, factor bag and spent media containerto the cultureware before running. The media and spent media containeris disconnected, pump cassette is unloaded, cultureware body is unloadedand the used cultureware is placed in a biohazard container fordisposal.

The purification unit of the integrated apparatus includes twoindividual parts: a reusable instrumentation base device (also referredto herein as a “second” reusable instrumentation base device, todistinguish it from the reusable instrumentation base device of the cellculture unit), and at least one disposable cell cultureware module thatis used for a single production run and is disposable (also referred toherein as a “second” disposable cell cultureware module, to distinguishit from the at least one disposable cultureware of the cell cultureunit).

The instrumentation base device of the purification unit provides thehardware to extract the fluid with the cell product from the cellculture unit and process it. An air detector checks the cultureware linewhich carries the fluid from the cell culture module to determine whenfluid is available to run through the column and when no more fluid isavailable. Drives for a plurality of switching valves (e.g., nineswitching valves) control the disposable valve portions to route fluidsto complete the processes. A peristaltic pump is used to move the fluidsto accomplish the process. A cooler lowers the disposable columntemperature to minimize product degradation. An optical density detectoris used in the process to determine when final product should becollected. The purification unit relies on the cell culture unit foruser interface and communications with the facilities data managementsystem. One or more pressure sensors may be included for monitoringfluid pressure for excessive pressures, or for control of peristalticpump speed, e.g., to maintain the pump speed at a desired pressure (afeedback mechanism). In some embodiments, the pressure sensor is placedin the purification flow path, on the output of the pumps.

In some embodiments, one or more correction sensors are employed formonitoring back-pressure. In some embodiments, one or more conductivitysensors may be used to monitor fluid exchange, and/or product harvest.In some embodiments, one or more optical density sensors are used tomonitor proteins in the fluid.

As is the case with the cultureware of the cell culture unit, thecultureware of the purification unit is for one-time use. The selectiondevice (e.g., purification column(s)) is loaded into the culturewarejust before use. The reservoirs are filled at that time with the correctbuffers for the cell product type. That information is tied to thecultureware's identifying code (e.g., bar code, radio frequencyidentification (RFID) tag, bokode, or quick response (QR) code) in thefacilities data management system when the operation is done and is usedto verify the proper purification cultureware is loaded for the cellproduct that is to be purified. A plurality of disposable switch valves(e.g., three disposable switch valves) are used to prepare thecultureware and route the fluids. An easy-load peristaltic pump cassetteis provided. A flow cell for measuring optical density is provided onthe outlet of the selection device. A removable container holds thefinished product (e.g., cell-derived product, such as antibody). Thepump cassette and cultureware body is unloaded from the instrumentationbase device of the purification unit and placed in a biohazard containerfor disposal.

As an enclosed apparatus, the safety provided by complete segregationfacilitates direct applicability to therapies or diagnoses that requireautologous cell culture. This self-contained, automated cell culture andpurification apparatus allows for simultaneous culture of numerous cellcultures within a compact facility, without the need for individual,segregated cell culture suites. The integrated apparatus of the presentinvention provides a compact, sealed containment apparatus that willenable the cost effective manufacture of cells, cell lines, cellproducts, including patient-specific cells and patient-specific cellproducts, and purification of the foregoing, on an industrial scale.

Another aspect of the present invention is to provide a method andapparatus that incorporates disposable cultureware, which eliminates theneed for cleaning and reuse.

Yet another aspect of the present invention is an apparatus that has thestand-alone integration of a large apparatus in a bench top device(pumps, controls, incubator, refrigerator, cultureware, etc.).

Still another aspect of the present invention is an apparatus thatincorporates an identification code reader, such as a barcode reader,and data gathering software that, when used with an informationmanagement system (such as a manufacturing execution system or MIMS),allows for automating generation of the batch record.

Another aspect of the present invention is to provide an EC cycling unitthat costs less than rigid reservoirs. Moreover, due to the sealed ECcircuit design, without vented reservoir, the chance of cellcontamination is minimized.

Still another aspect of the present invention is to provide an apparatusthat controls lactate concentration in a perfusion cell culture systemusing measurement of CO₂ and pH.

Yet another aspect of the present invention is to eliminate preparation,autoclaving, and insertion of pH electrodes aseptically in thecultureware which requires a significant amount of time and may breachthe sterile barrier of the cultureware set.

The apparatus of the present invention incorporates features thatgreatly reduce the operator's time needed to support the operations(e.g., integrated pump cassette, pre-sterilized cultureware with pHsensors, quick-load cultureware) and designed automated procedures andapparatuses which allow the apparatus to sequence through the operations(e.g., automated fluid clamps, control software).

The apparatus integrates the cell culture product production andpurification process. The design of the cultureware and instrumentsimplifies and reduces labor needed to produce product. This reducessources of error in the process.

The present invention provides an integrated, automated cell culture andpurification apparatus which creates a self-contained cultureenvironment. The apparatus incorporates perfusion culture with sealed,pre-sterilized disposable cultureware, such as hollow fiber or otherbioreactors, programmable process control, automated fluid valving, pHfeedback control, lactic acid feedback control, temperature control,nutrient delivery control, waste removal, gas exchange mechanism,reservoirs, tubing, pumps and harvest vessels. Accordingly, the cellculture unit (referred to as AutovaxID Cell Culture Module™) is capableof expanding cells in a highly controlled, contaminant-free manner.Cells to which this approach are applicable include transformed ornon-transformed cell lines, primary cells including somatic cells suchas lymphocytes or other immune cells, chondrocytes, myocytes ormyoblasts, epithelial cells and patient specific cells, primary orotherwise. Included also are cells or cell lines that have beengenetically modified, such as both adult and embryonic stem cells.Specifically, the automated cell culture apparatus allows for productionand harvest of cells or cell-secreted protein in a manner that minimizesthe need for operator intervention and minimizes the need for segregatedclean rooms for the growth and manipulation of the cells. Further, theapparatus provides a culture environment that is completelyself-contained and disposable. This eliminates the need for individualclean rooms typically required in a regulated, multi-use facility.Control of fluid dynamics within the bioreactor allows for growthconditions to be adjusted, e.g. changing growth factor concentrations,to facilitate application of unique culture protocols or expansion ofunique cells or cell lines. As a result, there is less variation andless labor required for consistent, reproducible production of cells forapplications to expansion of autologous cells and their use inpersonalized medicine applications.

According to these and other aspects of the present invention, there isprovided a cell culture unit for the production of cells and cellderived products including a reusable instrumentation base deviceincorporating hardware to support cell culture growth. A disposablecultureware module including a cell growth chamber is removablyattachable to the instrumentation base device.

According to these and other aspects of the present invention, there isalso provided a method for the production of cells and cell products ina highly controlled, contaminant-free environment comprising the stepsof providing a disposable cultureware module including a cell growthchamber, and a reusable instrumentation base device incorporatinghardware to support cell culture growth. The base device includesmicroprocessor control and a pump for circulating media through the cellgrowth chamber. The cultureware module is removably attached to theinstrumentation base device. Cells are introduced into the cell growthchamber. A source of media is fluidly attached to the culturewaremodule. Operating parameters are programmed into the microprocessorcontrol. The pump is operated to circulate the media through the cellgrowth chamber to grow cells or cell products therein. The grown cellsor cell products are harvested from the cell growth chamber. Thecultureware module is then disposed of.

In some embodiments, the cell products comprise immunoglobulin, such asIgM, IgG, IgD, IgE, IgA, or an immunoglobulin of mixed (chimeric)isotypes (e.g., IgM/IgD, IgM/IgA, IgM/IgG, or IgG/IgA; see, for example,Natsume A. et al., Cancer Res., 2008, 68(10):3863-3872).

In a particular embodiment, the present invention provides an automatedmethod of producing a vaccine by purifying a protein, such as anantibody (immunoglobulin), and conjugating the protein to an adjuvant,such as keyhole limpet hemocyanin (KLH). In some embodiments, theprotein is an antibody that targets a B-cell antigen expressed on B celltumors. In some embodiments, the antibody is an IgM, IgG, or mixedisotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the cell culture unit for producingcells and/or cell derived products according to the present invention.

FIG. 2 is another perspective view of the cell culture unit of thepresent invention.

FIG. 3 is a perspective view of the instrumentation device of the cellculture unit of the present invention.

FIG. 4 is a rear and partial side view of the instrumentation device ofthe cell culture unit of FIG. 3, with covers removed.

FIG. 5 is a rear view of the instrumentation device of the cell cultureunit of FIG. 3, with covers removed.

FIG. 6 is an enlarged view of the heating system of the instrumentationdevice of the cell culture unit of FIG. 3.

FIG. 7 is a perspective view of the variable output pump of the cellculture unit of the present invention.

FIG. 8 is an exploded view of the pump of FIG. 7.

FIG. 9 is a perspective view of the pump cassette of the apparatus ofthe present invention.

FIG. 10 illustrates the installation method of the cell culture moduleand instrumentation device of the cell culture unit of the presentinvention.

FIG. 11 is a perspective view of the gas blending and fluid cyclingcontrol of the module of the present invention.

FIG. 12 is a front view of the fluid cycling control of FIG. 11.

FIG. 13 is a perspective view of a rotary selection valve drive of thepresent invention.

FIGS. 14A and 14B are exploded views of the valve rotor of FIG. 13. andthe body used with it. FIG. 14C is a rear view of the valve body.

FIGS. 15A-15C are perspective views of a tubing slide clamp of thepresent invention

FIG. 16 is a perspective view of the factor and harvest bags of thepresent invention.

FIG. 17 is a perspective view of the disposable culture medium module ofthe present invention.

FIG. 18 is an interior view of the module of FIG. 17.

FIG. 19 is a perspective interior view of the back of the module of FIG.17.

FIG. 20 is a perspective view of the extra-capillary cycling unit of thepresent invention.

FIG. 21A is a flow diagram of the cycling unit of FIG. 20.

FIG. 21B is an exploded view of the cycling unit.

FIG. 22 is a flow diagram of the lactate control system of the presentinvention.

FIG. 23 is a flow diagram of the apparatus of the present invention.

FIG. 24 is another perspective view of the cell culture unit of thepresent invention.

FIG. 25 is a cross-sectional view of a flexible hollow fiber bioreactoraccording to the present invention.

FIGS. 26-31 are views of the touch screen associated with the automaticcontrol of the cell culture unit of the present invention.

FIG. 32 shows a schematic representation of an embodiment of thepurification unit, wherein the selection device, e.g., an affinitycolumn, and the diafiltration module of the purification unit areconnected to multiple liquid reservoirs. The reservoirs each containliquid, such as a wash buffers and an elution buffer for delivery to theselection device and acidic, basic and final buffer solutions fordelivery to the diafiltration module. Elution of the purified proteinfrom the selection device can be aided by a photometer. The apparatusfurther includes a device for flowing liquid from the reservoirs intothe selection device or into the diafiltration module, for example,valves and tubing which connect the reservoirs to the selection deviceor to the diafiltration module. The flow of the liquid is diverted byvalves which transfers the eluted protein-containing solution from theselection device to the diafiltration module and then to thepre-sterilized removable collection vessel (also referred to herein asthe second cultureware module).

FIGS. 33A and 33B are isometric and front views of an embodiment of theintegrated cell culture and purification apparatus of the invention,including the cell culture unit and purification unit adjacent thereto.

FIG. 34 shows an embodiment of a hollow fiber perfusion bioreactor ofthe Autovaxid instrument, with hollow fibers and in and out ports forthe intracapillary space and extracapillary space indicated. As will beappreciated by those skilled in the art, the sidedness and orientationof the ports on the bioreactor are not critical.

FIG. 35 is a flow diagram of an embodiment of the immunoglobulinpurification process.

FIG. 36 is an SDS-PAGE gel of cell free harvest (load) and finalproduct. Gel lane descriptions are listed in Table 3.

DETAILED DESCRIPTION OF THE INVENTION

The integrated apparatus of the invention includes two units: a cellculture unit and a purification unit (FIG. 33). The two units arephysically separate but designed to be placed together (e.g., adjacentto one another). The cell culture unit and purification unit cantransfer data and coordinate activity with each other using methodsknown in the art such as a communication port (e.g., an infraredcommunication port, desktop or laptop computer, etc.). The purificationunit can be placed next to the cell culture unit toward the end of theproduction period, or before. A tubing line from each unit's culturewareconnects together to provide a fluid path for the collected harvestfluid in the cell culture unit to be transferred to the purificationunit. The operator initiates the purification sequence through a userinterface such as a touch screen interface on the cell culture unit.Advantageously, the end product of the apparatus can be a purified andneutralized product in a buffered solution suitable for furtherprocessing.

Integrating components, functions, and operations greatly reducesmanpower and cost needed to produce a cell-derived product. Integratedcultureware reduces preparation and loading time. Culturewaresimplification reduces the number of operator induced errors which cancause failure. Process sequencing reduces operator time needed andallows sequential operations to be automatically. Modularizing thefunctions into a cell culture unit and a purification unit allows higherutilization of hardware and lower costs. One purification unit canservice multiple cell culture units. Utilizing the resources of the cellculture unit allows for reduced costs of the purification unit andlogistically ties the two processes together. A CGMP facility isrequired to generate a batch record documenting the individual cellproduct production. The apparatus can facilitate record generation byutilizing a central electronic records system operated in a CGMPfacility.

Some examples for which the apparatus of the present invention can beused are:

-   -   The production of monoclonal antibodies from hybridoma cell        lines.    -   The expansion of autologous patient-derived blood cells        including immune cells for therapeutic application.    -   The expansion of patient derived somatic cells for subsequent        re-infusion back into patients for therapeutic purposes. A        specific example already available for therapeutic application        in patients is the harvesting and expansion of patient specific        cartilage cells (chondrocytes) followed by re-infusion of those        cells back into a region containing damaged articular cartilage.    -   The expansion of patient derived or non-patient-derived        multipotent cells, including embryonic stem cells, adult stem        cells, hematopoeitic stem or progenitor cells, multi or        pluripotent cells derived from cord blood for therapeutic        purposes.    -   The expansion of somatic or germline cells in which the cells        have been genetically modified to express novel cellular        components or to confer on them other beneficial properties such        as novel receptors, altered growth characteristics or genetic        features, followed by introduction of the cells into a patient        for therapeutic benefit. An example is the expansion of patient        specific fibroblasts genetically modified to express growth        factors, clotting factors, or other biologically active agents        to correct inherited or acquired deficiencies of such factors.

In the methods and apparatus of the invention, harvesting can be carriedout by various methods, such as batch harvest, timed batch harvest, orcontinuous harvest. In batch harvesting, a single harvest of thebioreactor may be initiated by the operator based on product (e.g.,antibody) concentration. All product is collected at this time. In timedbatch harvesting, the harvest is initiated by the operator based onproduct concentration. A predetermined volume is harvested from thebioreactor. After a defined interval, another volume is harvested. Thisis repeated for a predetermined number of cycles or until the operatorterminates the harvesting. In continuous harvesting, the instrumentharvests a given volume per unit of time (e.g., hour) continuously. Theharvest can be initiated by the operator based on product concentration.Harvest continues until a time interval has passed or until the operatorterminates the harvesting.

Definitions

In order to more clearly and concisely describe the subject matter ofthe claims, the following definitions are intended to provide guidanceas to the meaning of specific terms used herein.

It is to be noted that the singular forms “a,” “an,” and “the” as usedherein include “at least one” and “one or more” unless stated otherwise.Thus, for example, reference to “a cultureware module” includes morethan one cultureware module, reference to “an affinity column” or “apurification column” includes more than one column, and the like.

As used herein, the term “adjuvant” refers to any substance whichenhances the immune-stimulating properties of an antigen.

The term “antibody,” as referred to herein, includes whole antibodiesand any antigen binding fragment (i.e., “antigen-binding portion”) orsingle chain thereof.

As used herein, the terms “automation” and “automated” are usedinterchangeably and refer to the controlled operation of an apparatus,process, or system by mechanical or electronic devices. Automatedmethods of the invention include sequential, pre-determined steps, whichare internally controlled by software driven servo-actuators. Thus, themethods are standardized, efficient and free of human error.

As used herein, the terms “comprising”, “consisting of” and “consistingessentially of” are defined according to their standard meaning. Theterms may be substituted for one another herein in order to attach thespecific meaning associated with each term.

As used herein, the term “computer system” generally includes one ormore computers, peripheral equipment, and software that perform dataprocessing. A “user” or “operator” in general includes a person, thatutilizes the system of the invention such as through a user interface. A“computer” is generally a functional unit that can perform substantialcomputations, including numerous arithmetic operations and logicoperations without human intervention. The apparatus and methods of theinvention can be computer-implemented via a computer system.

As used herein, “cultureware” refers to components which come in contactwith the cell product-containing aqueous medium (e.g.,protein-containing aqueous medium), the purified cell product (e.g., thepurified protein), or any liquid involved in the cell culture and/orpurification process. The purification cultureware (i.e., thecultureware of the purification unit) includes a pre-packed, disposable,pre-sanitized or pre-sterilized selection device, e.g., a column packedwith resin, which separates the protein from the contaminants containedin the protein-containing aqueous medium. The purification culturewarecan further include a pre-sanitized or pre-sterilized diafiltrationmodule which further serves to purify the protein, as well aspre-sanitized or pre-sterilized, disposable liquid reservoirs, valves,tubing, and collection vessels.

As used herein, the terms “diafiltration module” and “tangential flowfiltration cassette” are used interchangeably and generally refer tomembrane-based ultrafiltration devices. The diafiltration module workson the tangential flow filtration principle whereby molecules over50,000 daltons, such as proteins (e.g., antibodies such as IgG and IgM),cannot pass through the membrane but small molecules, such as buffers,can pass through. The diafiltration module is used to exchange onebuffer for another and is a more efficient substitute for dialysis. Inone embodiment, the diafiltration module contains a membrane havingabout 50 cm² area and a normal molecular weight limit or cutoff of50,000 daltons.

As used herein, the terms “pre-sanitized” and “sanitized” are usedinterchangeably and generally refer to components which have beencleaned to reduce the presence of contaminating substances and typicallypackaged (to remain sanitized), before use. Components which have beenpre-sanitized may also be pre-sterilized or sterile. As used herein, theterm “pre-sterilized” or “sterile” are used interchangeably andgenerally refer to components which are free from viable contaminatingorganisms and typically packaged (to remain sterile), before use.Accordingly, the pre-sanitized cultureware utilized in the presentinvention is free of contaminants which can contaminate the culture andpurification process, such contaminants can include viablemicroorganisms. Moreover, the method of the invention does not requirethe steps of sanitizing or sterilizing the cultureware to be used, sincethe cultureware is already or has previously been sanitized/sterilizedand is ready for use.

Still further, because the cultureware is disposable, the method doesnot require re-sanitizing or re-sterilizing components after use. Asused herein, the term “disposable” refers to components which aredesigned to be used and then thrown away. For example, the pre-sanitizedcultureware of the present invention can be designed to be used for asingle purification run and then thrown away. Accordingly, the presentinvention provides the advantage of eliminating the time-consuming andlabor intensive steps of pre-sanitizing or pre-sterilizing andpre-assembling the cultureware used to purify the biological product(e.g., protein).

By “solid phase” is meant a non-aqueous matrix to which the ligand canadhere, such as a solid phase comprising a glass or silica surface. Thesolid phase may be a purification column or a discontinuous phase ofdiscrete particles. In a particular embodiment, the solid phase is acontrolled pore glass column or a silicic acid column. Optionally, thesolid phase is coated with a reagent (such as glycerol) which preventsnonspecific adherence of contaminants to the solid phase. Affinityligands and methods of binding them to solid support materials are wellknown in the purification art. See, e.g., Affinity Separations: APractical Approach (Practical Approach Series), Paul Matejtschuk(Editor), Irl Pr: 1997; and Affinity Chromatography, Herbert Schott,Marcel Dekker, New York: 1997.

As further used herein, “tumor antigen” describes a polypeptideexpressed on the cell surface of specific tumor cells, e.g., anidiotypic tumor antigen expressed on the surface of B cells, and whichcan serve to identify the type of tumor. In some embodiments, theidiotypic tumor antigen is an IgM, IgG, or mixed isotype.

Cell Culture Unit

The cell culture unit of the integrated apparatus includes twoindividual parts: a reusable instrumentation base device (also referredto herein as a “first” reusable instrumentation base device, todistinguish it from the reusable instrumentation base device of thepurification unit), and at least one disposable cell cultureware modulethat is used for a single production run and is disposable (alsoreferred to herein as a “first” disposable cell cultureware module, todistinguish it from the at least one disposable cultureware of thepurification unit). The instrumentation base device provides thehardware to support cell culture growth and production in a compactpackage, which is advantageous in a facility handling a large number ofunique cell lines, for example. A pump, such as an easy-load 4 channelperistaltic pump, moves fresh basal media into the cultureware, removesspent media, adds high molecular weight factor and removes productharvest. An integrated cool storage area maintains the factor andharvest at a low temperature (preferably, approximately 4° C.). Aheating mechanism maintains the cell environment to promote growth andproduction. The gas blending mechanism, in conjunction with thecultureware pH sensor controls the pH of the cell culture medium. Aplurality of automated tube valving drives (e.g., three automated tubevalving drives) are used to control the cultureware flowpathconfiguration to accomplish the fluidic functions necessary to initiateand carryout a successful run. Valves and sensors in the instrumentationbase device control the fluid cycling in the cultureware. Drive forfluid circulation is provided. An identification code reader, such as abarcode reader, is preferably included to facilitate operator and lottracing. A communication port preferably ties the instrumentation deviceto a facilities data management system (LIMS). Preferably, theinstrumentation device of the cell culture unit includes a userinterface, such as a flat panel display with touch screen, for userinteraction.

The one-time use cultureware is provided pre-sterilized, designed forrapid loading onto the instrumentation base device (“quick-load”). Theloading of the cultureware body makes connections to the instrumentationbase device. The pump cassette, which is physically attached to thetubing, allows the user to quickly load the pump segments. The designand layout minimizes loading errors. The cultureware enclosure providesan area that is heated to maintain cell fluid temperature. Reservoirs tomaintain fluid volumes and cycling are included in the cultureware.Sensors for fluid circulation rate and pH and thermal well for theinstrument's temperature sensor are included. The blended gas from theinstrumentation device is routed to the gas exchange cartridge thatprovides oxygen and adds or removes carbon dioxide to the circulatedfluid to support cell metabolism. The cultureware module also includes abioreactor (e.g., hollow fiber bioreactor or other bioreactor type),which provides the cell space and media component exchange. Disposablecontainers for harvest collection and flushing are provided. Theoperator attaches a media source, factor bag and spent media containerto the cultureware before running. The media and spent media containeris disconnected, pump cassette is unloaded, cultureware body is unloadedand the used cultureware is placed in a biohazard container fordisposal.

Referring to FIG. 1, the present invention provides a fully integratedcell culture unit 10 for producing cells and cell derived products in aclosed, self-sufficient environment. More specifically, the cell cultureunit allows for cell expansion and harvest of cells and their productswith minimal need for technician interaction. As will be describedfurther herein, the device incorporates cell culture technology, forexample, hollow fiber or similar bioreactor perfusion technology, withall tubing components, harvest tubing and tubes threaded through thepump cassette, encased in a single-use, disposable incubator 12.Following bioreactor inoculation with cells, the apparatus followspre-programmed processes to deliver media, maintain pH, maintain lactatelevels, control temperature and harvest cells or cell-secreted protein.Standard or unique cell culture growth parameters can be programmed,such that, various cell types can be expanded and such that cells orcell products can be harvested in an efficient, reproducible manner withminimal chance of human error. The cell culture system and methoddescribed in International Publication No. WO 2007/139742, “Method andSystem for the Production of Cells and Cell Products and ApplicationsThereof” (Wojciechowski R. et al.), is hereby incorporated by referencein its entirety.

The cell culture unit is based on cell growth chamber technology. Forexample, bioreactors that have a plurality of semi-permeable hollowfibers or other type of semi-permeable membrane or substrate potted in ahousing to create a space inside the fiber or one side of the membrane(referred to as intracapillary or IC space) separate from that outsidethe fibers or on the other side of the membrane (referred to asextracapillary or EC space). Fluid distribution between the IC and ECspace occurs through the fiber pores which can range in size from 10 MW(Kd) to 0.2 μm. Cells are placed on one side of the fiber or membrane,usually in the EC space, in a complete cell culture medium, which isusually the same medium used to expand cells prior to bioreactorinoculation (serum containing, serum-free, or protein-free medium).Cells are usually placed in the EC space when secreted protein is thedesired product. In some instances, when cells are the desired product,it may be beneficial to place cells in the IC space.

Medium is perfused through a bioreactor 20 by circulating through the ICspace at a fast rate. The medium can be a liquid containing a welldefined mixture of salts, amino acids, and vitamins that often containone or more protein growth factors. This serves to deliver nutrients tothe cell space and conversely, removes or prevents a toxic build-up ofmetabolic waste. During this circulation, medium is passed through anoxygenator or gas exchanger cartridge 24 which serves to provide pHcontrol and oxygen for the cells and conversely, remove carbon dioxidefrom the culture. When the bioreactor 20 contains a smaller number ofcells, just after inoculation, the oxygenator or gas exchange cartridgeis used to provide CO₂ and subsequently control pH of the cultureenvironment. As cell number increases, the oxygenator is used to removeCO₂ which serves to enhance acid neutralization and control the pH ofthe culture. Other bioreactor configurations, in addition to hollowfibers, that are designed and optimized for the growth and production ofcells and production of cell-derived products may also be used.

The cell culture unit 10 provides significant efficiencies and costreduction through its disposable component and enclosed operation. Assuch, cells are contained in a closed system and continuously culturedwithout the need for specialized, segregated clean rooms. This fullyintegrated apparatus eliminates the need for cleaning and sterilizationvalidations, as well as the need for hard plumbing associated withconventional cell culture facilities.

Referring again to FIG. 1, the cell culture unit includes two individualparts: an instrumentation base device 14 that is reusable and anenclosed cultureware module 12 that is used for a single production runand is disposable. Numerous modules 12 can be used on a single device14. The instrument provides the hardware to support cell culture growthand production in a compact package. As shown in FIG. 2, and as will bedescribed in further detail herein, an easy-load multiple channelperistaltic pump drive 16 located in base device 14 and a pump cassette70 move fresh basal media into the cultureware, removes spent media,adds growth factors or other supplements and removes product harvest. Anintegrated cool storage area 18 maintains the factor and harvest at alow temperature (approximately 4° C.). An integrated heating mechanism22 (FIG. 6) maintains the cell environment to promote growth andproduction. Gas exchange cartridge 24 (FIG. 5), in conjunction with acultureware pH sensor 26 controls the pH of the cell culture medium. Twoautomated tube valving drives 90 (FIG. 3) are used to control thecultureware flow path configuration to accomplish the fluidic switchingfunctions needed to initiate and do a successful run. Valves 90 andsensors 32 (FIGS. 3, 5, 13) in the instrument control the fluid cyclingin the cultureware module 12. A pump drive 34 (FIGS. 3, 5) for fluidcirculation is provided. A wireless or tethered (attached)identification code reader (such as a barcode reader), shown in FIG. 33,facilitates operator and lot tracing. An identification code comprisesan identifier on or made part of a surface such as cultureware module oruser identification tag, and which may include, but is not limited to, abar code, a number, a series of numbers, a color, a series of colors, aletter, a series of letters, a symbol, a series of symbols, and acombination of one or more of the foregoing. Other examples includeradio frequency identification (RFID) tags, bokodes, and quick response(QR) codes. A communication port ties the instrument to a datainformation management system (such as a MES). A user interface 36, suchas a flat panel display (shown in FIGS. 1 and 33) with touch screencapability, is available for user interaction.

The one-time use cultureware module 12 of the cell culture unit isprovided pre-sterilized. It is designed for quick loading onto theinstrument (“quick-load”), as will be described further herein. Theloading of the cultureware body makes connections to the instrument.Pump cassette 70 (FIG. 2), which is physically attached to the tubing,allows the user to quickly load the pump segments. This design andlayout minimizes loading errors. The cultureware enclosure 12 providesan area that is heated to maintain cell fluid temperature. A fluidcycling unit 40 (FIGS. 1, 18) maintains fluid volumes and cycling and isincluded in the cultureware. Sensors for fluid circulation rate, pH anda thermal well for the instrument's temperature sensor are provided. Theblended gas from the instrument is routed to gas exchange cartridge 24that provides oxygen and adds or removes carbon dioxide to thecirculated fluid to support cell metabolism. A magnetically coupled pumpdrive 34 (FIGS. 11-12) circulates fluid thru the bioreactor 20 and gasexchange cartridge 24. The bioreactor 20 that provides the cell spaceand media component exchange is also in the cultureware. Disposablecontainers for harvest collection are provided. Prior to the beginningof the culture, the operator (also referred to herein as the user)attaches a media source, factor bag and spent media container to thecultureware before running. At the conclusion of the run the harvestcontainers are removed or drained, media and spent media container isdisconnected, pump cassette is unloaded, harvest bag disconnected,cultureware body is unloaded and the used cultureware is placed in abiohazard container for disposal.

Cell expansion and subsequent process tracking mandates generation of abatch record for each culture. Historically, this is done with apaper-based system that relies on operator input of the information.This is labor intensive and subject to errors. The fully integratedapparatus of the invention can incorporate an identification codereader, such as a barcode reader, and data gathering software which,when used with the information management system (MES), allows forautomatic generation of the batch record.

The apparatus of the present invention has application in a regulatedcell culture environment. It is anticipated that autologous whole celltherapies or patient-specific proteins (vaccines) therapies, would bytheir nature, require the simultaneous culture of numerous cell lines ina single facility. In addition to the segregation created through thisclosed culture approach, the apparatus is designed to support a standardinformation management system (such as a LIMS or MES) protocol. Thiscapability contributes to the creation of thorough batch records andverification of culture conditions to ensure standardization, trackingand safety of each product. This capability facilitates themulti-product concept that is pivotal to facilities involved withautologous or patient-specific products.

Referring to FIG. 1, disposable cell culture module 12 is removablyattachable to instrumentation base device 14. The module requiresmultiple mechanical and electrical interfaces to the controlinstrumentation of device 14. Module 12 has interface featuresintegrated into the module that mate with instrument interface featuresin the device to allow for a single motion installation (FIG. 10). Asmodules 12 are to be disposed of after use, it should be appreciatedthat numerous modules can be used in conjunction with a single basedevice 14.

As shown in FIG. 3, the interface features of device 14 includecirculation pump drive 34, actuator valves 90 and cycling sensor 32. Inaddition, a temperature probe 44 and a flow sensor 46 interface with thecomponents of module 12. Device 14 also includes an electricalconnection 48 for pH probe 26 disposed within module 12.

Gas ports 52 communicate with gas exchanger 24. One port 52 communicateswith the input to exchanger 24 and the other port 52 communicates withthe output of the exchanger. Gas ports 54 control pressure to thecycling fixture 40. One port 54 communicates with the IC chamber and theother port 54 communicates with the EC space. As viewed from the front,the left port 52 is the exchanger output and the right port 52 is theexchanger input. The top port 54 is the IC reservoir pressurizationport, and the lower port 54 is the EC reservoir pressurization port.

As described above, module 12 is heated to maintain cell fluidtemperature. Heating mechanism 22 (FIG. 6) maintains the cellenvironment to promote growth and production. The cell culture,disposable modules 12 requiring elevated temperatures are warmed byfully encapsulating the module and attaching the module to thecontrolling instrument device 14, such that air ports are aligned andwarmed air is forced into the module from the instrument at one locationand allowed to escaped at another. Instrument device 14 has a heated airoutlet 58 and a return heated air inlet 56.

When disposable module 12 is installed onto the controlling instrumentdevice 14, the air inlet 88 (FIG. 19) of the disposable module alignswith the air outlet 58 of the controlling instrument. Heating mechanism22 forces warmed air through outlet 58 and into the warmed air inlet 88and into disposable module 12. The warmed air elevates the temperatureof the components inside of the module. The exhaust air exits throughair outlet 86 and into air inlet 56 of instrument device 14 where it iscirculated.

During installation, module 12 is aligned with the connections of thedevice 14 and the module is placed into the operating position as shownin FIG. 10. All mating interface features are functional. Referring toFIG. 19, when installed, certain features of the module 12, formed in aback panel 148 of the module, interface with device 14. Module airoutlet 86 aligns with device air inlet 56 and module air inlet 88 alignswith device air outlet 58 to circulate heated air through module 12 asdescribed herein. Gas connectors 152 and 154 engage device gas ports 52and 54, respectively, to allow gas to enter and exit module 12. Valvebodies 156 receive actuator valves 90. Hub 158 receives pH probe 26interface and aligns with electrical connector 48. Module 12 isconnected to circulation pump drive 34 via module pump connection 164.Cycling unit 40 also communicates with cycling sensor 32 when the moduleis installed. The flow sensor 46 of device 12 mates with flow sensorconnection 166. The temperature sensor 44 of device 14 mates with a noninvasive receptacle in module 12 that is in contact with the IC media toprovide control feed back to the control mechanism to regulate thethermal output of heater 22. The above mating connections facilitate theone-motion installation of the module 12 on the device.

Referring to FIGS. 7-9, the present invention incorporates amulti-position, cassette loading, and peristaltic pump 16 (FIG. 2) withdiscrete, variable output control for each channel. A plurality ofchannels 60 (FIG. 3) are located in device 14. Although four channelsare shown, it should be appreciated that pump 16 could have more or lesschannels.

As shown in FIG. 8, the pump has individual, variable control of theoutput of each channel. Pump rotors 62A-62D have a common fixed axialshaft 64 with individual servo drive. The occlusion rotors 66A to 66Dare mounted to the pump rotors 62A-62D, which in turn are mounted on thesingle shaft 64 with internal bearings that allow for independentfunctional control by a respective reacting servo drive 68A-D. Thesingle shaft minimizes tolerance accumulations typically caused bymisalignment of individual rotors and shafts mating with a multi-channelcassette. Feedback sensors are included to verify rotation of the pumprotors.

Typical multi-channel peristaltic pump applications operate using arotating drive shaft that is common to all rotors. This causes allrotors to turn at the same revolution per minute (RPM), yielding thesame fluid output. Different inside diameter tubing may be used to givea fixed ratio delta output from one rotor to another. To obtain avariable output of the peristaltic pump segments, individual pump headsand drives are used. This requires individual tubing cassettes that mustbe loaded individually and does not allow for close center to centerdistance between pump heads.

As shown in FIG. 9, a multi-channel cassette 70 is featured withpre-loaded peristaltic tubing 72 to reduce loading errors and to reduceinstallation time. The mechanism includes a cam operated cassetteinsertion feature 74) that interfaces with 67 on pump 16. As shown inFIG. 8, a knob 65 is rotated to move cam feature 74 into position to aidinitial tubing occlusion during loading.

The cassette configuration is structured to hold multiple peristaltictubing segments. A gripping feature 76 on the top and the bottomprevents the tubing from creeping during operation. The design allowsfor all tubing segments to be loaded into the pump drive mechanism atthe same time. A latching feature 74 is also included to provide abearing surface for the cam-operated latch 67 to react upon.

Referring back to FIGS. 1 and 2, cassette 70 is pre-loaded withperistaltic tubing (FIG. 24) and positioned in groove 80 on module 12.After module 12 is positioned on device 14, cassette 70 is removed andinserted into interface or plate 82. Each cassette section 71 (FIG. 9)supporting the tubing is inserted into a respective channel 60 (FIG. 3)of the interface 82. This configuration reduces tubing segment loadingerrors with pre-loaded multi-position cassettes, and reducesinstallation time.

Referring to FIGS. 11 and 12, valves and sensors 32 in the instrumentcontrol the fluid cycling in the cultureware module 12. Two opticalsensors detect the low or high position of the cycling position sensorflag 140 (FIG. 20). This information is used by a predictive algorithmto control the pressures applied to the IC chamber and EC pressure bagto effect cycling.

Sterilizable, disposable, actuator driven, rotary selection valves 90are shown in detail in FIGS. 13-14C. Valve 90 comprises a valve housing93 and valve cam 94. The elastomer tubing (not shown) is insertablethrough openings 98 in valve body 92 and is occluded by a rotating cam94 that compresses the tubing against the valve body. This isaccomplished by using a controlled, incremental, servo drive 96(actuator and position feed back loop) to move cylindrical cam thatreacts against immobile valve body 92 that holds the tubing in aconstrained state. The cam design allows for a high area of the cam 104to occlude the tubing and a low area of the cam 106 not to occlude,resulting in a closed and open condition respectively. Cam rotationalpositioning features may also be added to move cam 94 to predeterminedpositions. Configurations can be structured to accommodate multipletubing segments in one device. The two piece design allows for fluidcontact portion of the valve to be molded into the backpanel 148 (FIG.19) as a hub 156 and to be sterilized (EtO, chemical or radiation) withthe rest of the fluid circuit and eliminates the need to be addedseparately.

The design of this clamp is meant to be used in an automated cellculture application where a disposable cultureware module interfaceswith an electro-mechanical instrument. The combined unit is to beautomated, which required various tubing lines of the disposable to beoccluded/open to provide automated process control. The selector valveis used to automatically open and close tubing lines to direct fluid orgas flow during process control. Minimizing operator set-up is also arequirement. The disposable cultureware must be inserted into theinstrument in an operating position with no special operator proceduresrequired for loading the tubing into the clamps. Existing technologiesdid not meet these requirements, because the manual clamps were notautomated, and solenoid valves required a special operator loadingprocedure.

In the cell culture and purification units of the present invention, thefluid path must be free of unwanted organisms (sterilized). Commerciallyavailable selector valves are not gas sterilizable. Sealing surfaces ofthe selected position may be unexposed to the gas sterilant and thosesurfaces may be “non-sterile” when the valve is repositioned. Valve 90provides automated actuation of the cam, compactness, multiple lines,maintains valve position even with loss of actuator power, thedisposable valve body is less costly than an equivalent switching valve,and can be incorporated into the back panel of 12. Offset occluded/opencam positioning of two tubing lines can insure a make-before-breakswitching of fluids. No power is required to maintain any operatingposition, and tubing segments used in the valve body can be sterilized.

It should be appreciated that a solenoid driven pinch mechanism, can beused in place of the actuator valve. This application may utilize apiston plunger actuated by an electrical coil to provide linear motionto pinch the tubing. A manual pinch clamp could also be used. Theclamping position is manually activated by a mechanical bearing surfacecompressing the tubing and then held in position by a detent feature.This clamp type requires manual deactivation. A membrane over the seriesof ports could also be used. The membrane is actuated against the portto seal it. Multiple ports are configured for use as a selectormechanism.

In another embodiment shown in FIGS. 15A-15C, an actuator driven tubingslide clamp 110 with multiple positions and multiple tubing can be usedas an alternative to valves 90. Elastomer tubing is occluded by slidingthe tubing into a narrow slot 112 that compresses the tubing wallagainst itself. This is accomplished by using a servo drive (actuatorand position feed back loop) to move a plate 110 with slot 112 in it andreacting against another plate or slide body 114 that holds the tubingin an immobile state. The moveable plate is designed with varying widthslots to allow for position/positions to be inactive. This allows fornormally open 116 or 118 and normally closed 117 positions.Configurations can be structured to accommodate multiple tubing segmentsin one clamp.

In operation, slide 110 is positioned into slide body 114. Tubing isinserted through tubing ports 108 and slide 110 at position 116 whereboth tubes are not occluded. A remote servo (not shown) engages intoserver drive slot 102 and moves the slide to position 117 where one tubeis occluded and one tube is not occluded. The remote Servo than movesthe slide to position 118 where the occluded tube from the previous stepis not occluded, and the tube from the not occluded tube from theprevious step is now occluded. When moving the slide from position 117to position 118, both tubes are occluded to insure that one tube isoccluded before the other tube is opened. It should be appreciated thatthe number of tubes and configuration of the slide can be modified tomeet customized applications.

The clamp is meant to be used in an automated cell culture applicationwhere a disposable cultureware module interfaces with anelectro-mechanical instrument. The combined unit is to be automated,which required various tubing lines of the disposable to beoccluded/open to provide automated process control. During processcontrol the clamps are open/closed to simulate the function of anexpensive, “disposable” switching valve. Minimizing operator set-up isalso a requirement. The disposable cultureware must be inserted into theinstrument in an operating position with no special operator proceduresrequired for loading the tubing into the clamps. It provides automatedactuation of slide clamp, compactness, multiple lines, maintains clampposition even with loss of actuator power, less costly than anequivalent switching valve. Offset occluded/open position of two tubinglines can insure a make-before-break switching of fluids. No powerrequired to maintain any operating position.

As described above, integrated cool storage area 18 maintains growthfactors and harvested cells or cell products at a low temperature(approximately 4° C.). Referring to FIGS. 2 and 16, a rack 120 isremovably positionable within cool storage area 18. Rack 120 is designedto support a plurality of bags 122, 124. The bags are used to containthe smaller quantities of product or growth factors. It should beappreciated that other solutions can be disposed with the bags. Forexample, high molecular weight growth factor can be located with bag122. This factor is connected via tubing 128 to the bioreactor or cellgrowth chamber 20 and the flow controlled by pump 16. Harvested cells orcell products can be stored in bag 124. A cell filter 126 is provided toprovide additional filtration. A filter bypass line is included iffiltering of the harvest is not desired as in the case of cellcollection. After the process is complete the cells can be removed fromthe cell culture chamber via the tubing and stored in bag 124 until use.

As shown in FIGS. 17-19, disposable cultureware module 12 includes fluidcycling unit 40 to maintain fluid volumes and cycling in the cell growthchamber. Referring to FIGS. 18-21, the present invention utilizesextra-capillary (EC) cycling in cell culture growth chamber 20 (FIG. 17)utilizing a non-rigid, EC reservoir 130 and mechanical or a secondflexible reservoir 132 to cause elevated EC pressure. Reservoirs 130,132 are separated by a sensor plate 134. Reservoirs 130, 132 arerestricted in the maximum amount of expansion by a rigid mechanicalhousing 136. EC cycling is achieved by utilizing a non-rigid reservoirto retain the varying fluid volume associated with an EC circuit.Flexible reservoir 130 is fluidly connected to the bioreactor or hollowfiber device 20. Second flexible reservoir 132 is pressurized to applyforce against the flexible reservoir 130 to provide an elevated ECpressure to cause an ultra-filtrative condition and force fluid into anintra-capillary (IC) circuit 138. A mechanical feed back positionindicator 140 is physically connected to sensor plate 134 and moves withthe physical expansion and contraction of the first flexible reservoir.The position of indicator 140 is sensed by the position sensors 32 andis used to control the force that is applied by second flexiblereservoir 132. It should be appreciated that an alternate mechanicalforce apparatus may be used instead of a second flexible reservoir tocause pressure changes.

During operation the pressure is increased in the IC circuit 138 bypressurizing an IC reservoir 137. This pressure causes anultra-filtrative condition that forces fluid transmembrane across thesemi-permeable matrix of the bioreactor 20. The fluid is then forcedthrough the connect tubing, through a flow control valve 133 and intothe EC reservoir 130. Externally controlled pressure in the pressurereservoir 132 is allowed to vent. The expanding EC reservoir 130 forcesthe sensor plate 134 toward the pressure reservoir 132 and compressesit. Sensor plate 134 moves external position flag 140 and this is sensedwhen EC reservoir 130 has filled enough to expand to the EC upper level.The external position sensor 32 senses this position and the pressure inthe IC reservoir 137, is decreased and the pressure in the pressurereservoir 132 is increased. This causes an ultra-filtrative conditionand forces fluid out of the EC reservoir through a control valve 135,transmembrane across the matrix of the bioreactor 20 and into the ICcircuit 138. The sensor plate 134 moves the external position flag 140and the sensor 32 senses when the EC reservoir 130 has contracted to theEC low level.

The EC cycling unit of the present invention offers fluid dynamics tocause fluid flow in the EC space thus minimizing nutrient and metabolicwaste gradients that may be detrimental to the cells. It provides fluidlevel control without the use of ultrasonics or load cells that is notaffected by cell debris. The flexible reservoirs are considerably lessexpensive and are suited for disposable applications. The sealed ECreservoir with cycling also limits contamination and isolates the cells.

The present invention also includes an indirect lactate control methodfor perfusion culture using CO₂ and pH sensing. The method predicts opensystem, perfusion culture, lactate levels in the circulatory medium bymonitoring the pH and off-gas CO₂ level. This is accomplished bycalculating the initial bicarbonate level of the media then utilizingthe liquid pH and gas level of CO₂ to calculate current lactateconcentration. This is used to control media dilution rate of the cellculture. The resulting calculated lactate value is used to set theperfusion rate of media dilution to maintain a pre-determined lactatelevel. Thus, an invasive sensing system or multiple off-line sampling isnot required.

A physical relationship exists between bicarbonate buffer, dCO2, and pH.pH=pK+log([HCO₃ ⁻]/dCO_(2]))  Equation (1):

where:

-   -   pH=the pH of the solution    -   pK=the acid ionization constant for bicarbonate    -   HCO₃ ⁻=the current bicarbonate concentration (mM)    -   dCO₂=the concentration of dissolved CO₂

Lactic acid production by the cells appears to be the dominant drivingforce for pH changes in cell culture media. Based on this observation,each mole of lactic acid produced results in consumption of one mole ofbicarbonate as described by the following equation:[HCO₃ ⁻]═[HCO₃ ⁻]₀−[Lactate]  Equation (2)

where:

-   -   [HCO₃ ⁻]₀=the initial bicarbonate concentration in the medium        (mM)    -   Lactate=the lactate concentration (mM)

Equation (3) provides a simple relationship—Henry's Law, thatequilibrium dCO₂ is proportional to the gas phase concentration of CO2.dCO₂ =a(% CO₂)  Equation (3):

where:

-   -   a=CO₂ solubility conversion (mM/%)    -   % CO₂=concentration of CO₂) in the gas phase that is in        equilibrium with dCO₂ (%).

Equation (4) is derived by substituting Equation 2 in Equation 1 asfollows:pH=pK+log {([HCO₃ ⁻]₀−[Lactate])/[dCO₂]}  Equation (4)

Equation 5 is derived by combining Equations 3 and 4:pH=pK+log {([HCO₃ ⁻]₀−[Lactate])/[a(% CO₂)]}  Equation (5)

The operating equation, Equation (6) is derived by solving for Lactatein Equation (5):Lactate=[HCO₃ ⁻]₀−(a)*(% CO₂)*10^((pH-pK))  Equation (6):

The values of pK and (a) were found to be 6.38 and 0.39, respectively.

Upon taking a lactate and pH reading, the value of (a) is calculated.The initial bicarbonate concentration is calculated as the calibrationconstant. The advantage is that the bicarbonate concentration does nothave to be known when using the present calibration method.

The application is shown in FIG. 22. In a bioreactor perfusion loop, thegrowth media is pumped from an IC reservoir 137 via pump drive 34, 164,circulated to the gas exchange cartridge (GEX) 24, pH sensor 26,bioreactor 20, and then back to reservoir 137. Blended gases are passedthrough the membrane gas exchange cartridge that oxygenates the mediaand regulates CO₂. Per Henry's Law, the CO₂ levels in the gas phase orair side of the GEX 24 is in equilibrium with the liquid phase of themedia. The discharge end of the GEX is monitored with a CO₂ sensor 142that resides in the device 14 and the lactate is calculated per Equation(6). When the media lactate level is known, the instrument usesautomatic, media dilution, control to maintain the predetermined setpoint.

The present invention utilizes existing signals and with the addition ofa non-invasive gas CO₂ sensor incorporates lactate control to controlmedia feed rate for cell growth and production. Utilizing the inventionreduces materials and labor associated with recurring off-line testing.Utilizing the invention allows for continual adjustment of the dilutionrate that would otherwise be inefficient and costly if step increaseswere used as in previous technologies.

Utilizing the present invention increases the predictability of cellculture metabolics and allows a perfusion cell culture unit to have anincreased level of automation. The lactate and media dilution rate canbe used to determine the state of cell growth and production.

The present invention also utilizes a novel approach for pH sensing in acell culture unit. Referring back to FIGS. 2, 18 and 19, pH probe 26 anda holder are built into cell culture disposable 12, thus the user is notrequired to add the probe to the cultureware. Probe 26 is intended to bea one-time use device that is disposed of with the cultureware. Theprobe is disposed of with the used cultureware, no time is spentrecovering the probe for cleaning, revalidating and reuse.

In operation, the probe 26, for example, a solid gel filled electrode,is mounted in a holder 28 (FIG. 23) through which the media to be sensedflow. The electrode in the holder is fluidically connected to thecultureware circuit, mounted in the cultureware module, the circuit ischecked for fluidic integrity, and sterilized with the completedcultureware (ethylene oxide, EtO). After sterilization, QC checks areperformed on the EtO process to provide high confidence ofsterilization. When an operator wishes to culture cells, the culturewareis removed from the pouch, loaded on the instrument and fluid isintroduced into the cultureware. A period of time is given to re-hydratethe electrode. The cultureware is brought to operating conditions, theelectrode is calibrated and then used to control pH in the cultureware.When the cell culture is complete, the operator disposes of thecultureware and the probe. Although the probe has been described as asolid gel electrode other probe types could be used (e.g., an ISFET,liquid filled, immobilized phenol matrix, fluorescence, etc.).

Referring to the flow diagram of FIG. 23, pump 16 moves fresh basalmedia into the cultureware at media line 210. Media line 210 isconnected to a user provided container of fresh media to provide thegrowth nutrients to the cell culture that are pumped into thedisposable. Outflow line 214 is connected to a user provided containerto collect the waste or spent media being pumped out of the disposable.Factor line 212 is connected to a user provided container of growthfactors that are pumped into the disposable. EC inoculate can be addedat 220 and IC sample at 216. Product harvest is removed at 126. Thecells are harvested at 218. Harvest line 218 is a pre-attached containerthat is part of the disposable that is used to collect the product thatis pumped out of the disposable. Pump 16 has multiple lines 210, 214,212 and 126. Because the pump of the present invention has a commonfixed axial shaft and individual servo driven rotors, the control of theflow of each can be independent, allowing one channel or flow to beincreased while another decreased.

As shown in FIG. 25, a bioreactor 170 may have a flexible outer body 172allowing for physical movement of the cell growth substratum (hollowfibers, membrane or other suitable matrix) when a resultant torqueing orbending moment is applied to the bioreactor ends. Flexible outer body172 allows for the bioreactor case to be flexed causing fiber movement.This fiber movement enhances the release of cells that have attached tothe side of the bioreactor matrix. The cells can then be harvested byflushing either after or during the manipulation. This method canprovide increased efficiency of cell harvest at high cell viabilitieswithout the use of chemical or enzymatic release additives.

A bioreactor can be constructed using an outer housing that incorporatesa flexible center section. This center section is composed of aflexible, non-permeable tubing that allows each end of the bioreactor tobe manipulated, thus causing movement of the growth matrix. The purposeof this movement is to release the attachment or clumping of cellproducts on the extra-capillary (EC) side of the fibers. The cellproducts can then be flushed from the EC via the access port at each endof the bioreactor.

Harvesting cells from a matrix-containing bioreactor such as a hollowfiber bioreactor has been difficult to accomplish. Typically, cells aresticky and attach themselves to the fibers or to other cells and formclusters. Rapid flushing of media through the EC to hydraulically forcethe cells free and into the harvest stream is the most basic method ofharvesting cells from the EC space. Typically the quantity of cellsharvested is low because the flushing media tends to shunt through theEC and flush cells only from the limited fluid path.

Another method is to physically shake or impact the outer housing torelease the cells or clumps of cells. This practice may cause physicaldamage to the bioreactor or its associated components. Another methodincludes the use of chemicals to disrupt the adhesion of cells to thefibers or to disrupt the clumps of cells. Adding chemicals to acontrolled process may cause adverse effects on cell viability and canintroduce an unwanted agent in the down-stream processing.

Referring to FIGS. 26-31, various views of the touch display screenillustrate the different interactive steps during control process of theapparatus of the present invention. FIG. 26 shows a system overviewscreen which highlights current conditions. FIG. 27 illustrates a runsequence screen which directs the operator through the culture process.FIG. 28 illustrates log data which the operator can review and which isavailable to build the batch record. FIG. 29 shows a method forinputting alpha-numeric data. FIGS. 30 and 31 show operator interactionscreens to assist in operations (factor addition and pH probecalibration). On line help screens aid the operator for correctoperation.

The apparatus of the present invention fully integrates the concept ofdisposable cultureware into automated process control for maintainingand expanding specialized (autologous or other) cell lines for aduration of any time needed. To accomplish this, the apparatus of thepresent invention was designed for EC space fluid flow that enhancescell growth in high density perfusion culture, yet remains completelyclosed and disposable. The integrated pre-assembled cultureware, whichconsists of all tubing, bioreactor, oxygenator, pH probe, is enclosed ina single unit that easily snaps into the apparatus. In addition to thiserror-proof, quick-load design, the entire cultureware unit enclosed bythe casing becomes the cell culture incubator with temperature controlregulated through automated process control of the instrument. Pumps andfluid control valves facilitate disposability and error-proofinstallation, eliminating the possibility of technician mistakes.Finally, during the course of any culture, as a closed system,restricted access is facilitated, except for trained and authorizedpersonnel. Manipulations or sampling, outside of program parameters, canrequire password and identification code (e.g., bar code radio frequencyidentification (RFID) tag, bokode, or quick response (QR) code) accessbefore they can be implemented.

Purification Unit

The purification unit of the integrated apparatus is an automatedapparatus for obtaining a purified biological product such as protein(e.g., a purified antibody), from a biological product-containingaqueous medium (e.g., protein-containing aqueous medium). Thepurification unit includes two individual parts: a reusableinstrumentation base device (also referred to herein as a “second”reusable instrumentation base device, to distinguish it from thereusable instrumentation base device of the cell culture unit), and atleast one disposable cell cultureware module that is used for a singleproduction run and is disposable (also referred to herein as a “second”disposable cell cultureware module, to distinguish it from the at leastone disposable cultureware of the cell culture unit).

The instrumentation base device of the purification unit provides thehardware to extract the fluid with the cell product from the cellculture unit and process it. An air detector checks the cultureware linewhich carries the fluid from the cell culture module to determine whenfluid is available to run through the column and when no more fluid isavailable. Drives for a plurality of switching valves (e.g., threeswitching valves) control the disposable valve portions to route fluidsto complete the processes. A peristaltic pump is used to move the fluidsto accomplish the process. A cooler could be used to lower thedisposable column temperature to minimize product degradation. Anoptical density detector is used in the process to determine when finalproduct should be collected. The purification unit relies on the cellculture unit for user interface and communications with the facilitiesdata management system. The purification apparatus and method describedin International Publication No. WO 2005/090403, “Method and Apparatusfor Protein Purification” (Gramer M. et al.), is hereby incorporated byreference in its entirety.

As is the case with the cultureware of the cell culture unit, thecultureware of the purification unit is for one-time use. The selectiondevice (e.g., a purification column) and diafiltration module are loadedinto the cultureware just before use. The reservoirs are filled at thattime with the correct buffers for the cell product type. Thatinformation is tied to the cultureware's identifying code (e.g., barcode, radio frequency identification (RFID) tag, bokode, or quickresponse (QR) code) in the facilities data management system when theoperation is done and is used to verify the proper purificationcultureware is loaded for the cell product that is to be purified. Aplurality of disposable switch valves (e.g., seven disposable switchvalves) are used to prepare the cultureware and route the fluids. Twoeasy-load peristaltic pump cassettes are provided. A flow cell formeasuring optical density is provided on the outlet of the purificationcolumn. A removable container holds the finished product (e.g.,cell-derived product, such as antibody). The pump cassette andcultureware body is unloaded from the instrumentation base device of thepurification unit and placed in a biohazard container for disposal.

The purification unit utilizes pre-sanitized or pre-sterilizeddisposable cultureware, such as pre-sanitized or pre-sterilizeddisposable selection device, diafiltration module, liquid reservoirs,valves, tubing, and collection vessels, which can be packaged togetherfor single use and then disposed of. Accordingly, the purification unit(also referred to as an Autovaxid Purification Module) is capable ofpurifying proteins, such as antibodies, in a highly efficient andcontaminant-free manner. Specifically, the automated purification unitminimizes the need for operator intervention and provides a completelydisposable flowpath to eliminate the need for cleaning and to eliminatethe potential for cross-over contamination. Therefore, the purificationunit is an automated apparatus for purifying proteins and other cellproducts in a less labor intensive manner compared to manualpurification methods, thus, reducing purification time and increasingefficiency.

In one embodiment, the purification unit has the configuration shownschematically in FIG. 32. The purification unit comprises apre-sanitized or pre-sterilized, disposable cultureware module, asdiscussed above. With reference to FIG. 32, the cultureware moduleincludes, for example, a selection device (e.g., a purification column),a diafiltration module, multiple liquid reservoirs, a device for flowingliquid from the reservoirs and into the selection device and thediafiltration module, a device for diverting the effluent from theselection device and the diafiltration module, e.g., at least tworeservoirs. The cultureware module of the purification unit is capableof being installed into the instrumentation base device via a singlemotion or “snap-on” or “quick-load” technique and comprises mechanicaland electrical interfaces for communicating with the instrumentationbase device.

In the diafiltration loop (mixing chamber, pump, pressure guage and TFFcartridge), there is a volume of fluid. Because of the physicalcharacteristics of the TFF cartridge, molecules above a certain size areretained in the loop, smaller molecules can flow (permeate) across thefiber and out the permeate port of the TFF device. Pressure on the loopside determines rate of flow across the membrane for a specificmembrane. Pressure is generated by flowing fluid through the TFF and/orrestricting the outlet tubing of the TFF device returning fluid to themixing chamber. Because the TFF loop is a closed loop, permeating fluidcauses a negative pressure in the head space of the mixing chamber. Thisprovides the hydrostatic pressure needed to draw fluid from the selectedbuffer bag to make up the volume. Due to the small volume in the loop itis efficient at buffer exchange (it takes less fluid to get to a desiredconcentration or reduction than dialysis).

Permeate of diafiltration may be monitored by weighing collectedpermeate fluid. The permeate rate for a given pressure will change overthe course of the diafiltration due to membrane fouling. With that inmind, by observing the amount of fluid that has permeated from the loop,an accurate prediction of the loop concentration can be made. Since itis easier to measure weight than volume aseptically and knowing 1 gramof fluid is approximately 1 ml, incorporating a scale system (“scale” inFIG. 32) which captures the permeate is a viable system for controllingthe buffer exchange process. As an example, if a buffer exchange isdesired that will reduce the current buffer to 2% of the originalconcentration with a different buffer in a 20 ml loop volume, permeating100 ml while adding the new buffer would provide the desired result(exchanging 5 loop volumes).

With reference to FIG. 32, the apparatus may include two viral reductiondevices: a virus filter and an anion exchange filter. The anion exchangeis a host cell product and viral reduction device. For tangential flow(TFF), Millipore Pellicon XL Biomax 50 TFF cartridge may be used toprovide buffer exchange. This cartridge contains a 50 cm²polyethersulfone (PES) membrane, with a molecular weight cut off of 50kilodaltons (kD). This filter will retain molecules that are larger than50 kD in size while allowing smaller molecules to pass through. Thismechanism allows for control of the concentration of the product byincreasing or decreasing the volume of the protein containing solutionretained by the filter as well as providing a mechanism for exchangingthe buffer components of the protein solution. For anion exchange, aBio-Rad Bio-Scale Mini Unosphere Q Cartridge (#732-4101) may be used toprovide anion exchange chromatography (AEX) capability. Anion exchangeexploits differences in charge between the product and impurities. Theneutrally charged product passes over the AEX cartridge in flow-throughmode, while negatively charged impurities are retained.

For the virus filter, an Asahi Kasei Planova Viral Filter (EXZ-0010) maybe used to filter the final product. The virus filter is specificallyfor removing viruses from solutions containing biological molecules. Inthis case, virus filtration works on the principle of size exclusion.Planova filters contain a bundle of straw-like hollow fibers. When aprotein solution with possible viral contamination is introduced intothese hollow fibers, the smaller proteins penetrate the fiber wall andworks its way to the outside of the fiber while the larger virusparticles are retained. This may be used for IgG production (IgM willnot cross the fibers).

In a particular embodiment, the selection device, e.g., an affinitycolumn, and the diafiltration module of the purification unit areconnected to multiple liquid reservoirs. The reservoirs each containliquid, such as a wash buffer, an elution buffer, or a neutralizationsolution, for delivery to the selection device or the diafiltrationmodule. Accordingly, the purification unit further includespre-sanitized or pre-sterilized device for flowing liquid from thereservoirs into the selection device, for example, pre-sterilized valvesand tubing which connect the reservoirs to the selection device. Thevalves and tubing may allow liquid from only one reservoir at a time topass through the selection device. Alternatively, the valves and tubingallow for liquid from more than one reservoir to pass through theselection device.

In a particular embodiment, the purification unit includes apre-sanitized or pre-sterilized device for diverting the effluent fromthe selection device into the diafiltration module or into a wastecontainer. Similarly, the purification unit includes a pre-sanitized orpre-sterilized device for diverting the effluent from the diafiltrationmodule into the pre-sterilized collection vessel or into a wastecontainer.

In one embodiment, the purification method of the invention begins withthe automated step of loading a protein-containing aqueous medium, e.g.,an antibody-containing aqueous medium, onto a pre-sanitized, preferablya pre-sterilized, disposable selection device to absorb the protein ontothe selection device. The selection device for use in the presentapparatus and method can include, for example, a column packed with anaffinity resin, such as an anti-IgM resin, a Protein A, a Protein G, oran anti-IgG resin. In another embodiment, the protein-containing aqueousmedium and the selection device can be pre-treated prior to loading. Forexample, the protein-containing aqueous medium can be automaticallyheated and degassed before loading. The selection device can be washed,pre-eluted, and/or pre-neutralized prior to loading.

After loading, the selection device is typically washed to removeresidual contaminants contained within the aqueous medium, such asresidual proteins from host cells used to produce the protein to bepurified, e.g., host cell proteins, nucleic acids and endotoxins.

After washing, the bound protein is then eluted into an aqueous medium.The step of eluting can be accomplished, for example, by either changingthe pH or the salt concentration of the solution which is loaded ontothe selection device. For example, an acidic solution can be added tothe selection device to produce a protein-containing acidic eluate. Anexample of an appropriate acidic solution for eluting includes asolution of approximately 0.05 to 0.5 M of an acid (such as, glycine orcitrate) at a pH of about 2 to 5. Alternatively, the step of eluting caninclude adding a solution to the selection device which alters the saltconcentration of the aqueous medium loaded onto the selection device. Inone embodiment, the step of eluting the protein is facilitated by theuse of a photometer.

Upon eluting the protein into an aqueous medium, the eluted purifiedprotein can be automatically deposited into a pre-sterilized, disposablecollection vessel and removed from the automated purification apparatus.Alternatively, the eluted purified protein can undergo further automatedprocessing.

In one embodiment, the eluted purified protein, e.g., an antibody, istransferred to an acidic solution. This transfer to an acidic solutioncan be accomplished by using, for example, a diafiltration modulecontained within the automated apparatus. The diafiltration module is amembrane-based ultrafiltration module installed within the automatedpurification unit which utilizes the tangential flow filtrationprinciple. Accordingly, the eluted protein is diafiltered against anacidic solution (e.g., a solution of approximately 0.1 M glycine at a pHof about 2.0 to 5).

The method of the invention can include the step of placing the elutedprotein in contact with the acidic solution for approximately less than16 hours to inactivate any susceptible virus that may be containedwithin the solution. For example, the protein is held in the acidicsolution for approximately 15.5 hours, 15 hours, 14.5 hours, 14 hours,13.5 hours, 13 hours, 12.5 hours, 12 hours, 11.5 hours, 11 hours, 10.5hours, 10 hours, 9.5 hours, 9 hours, 8.5 hours, 8 hours, 7.5 hours, 7hours, 6.5 hours, 6 hours, 5.5 hours, 5 hours, 4.5 hours, 4, hours, 3.5hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour or less. Inanother embodiment, the protein is held in the acidic solution for lessthan 1 hour, for example for approximately 55 minutes, 50 minutes, 45minutes, 40 minutes, 35 minutes, 30 minutes, 29 minutes, 28 minutes, 27minutes, 26 minutes, 25 minutes, 24 minutes, 23 minutes, 22 minutes, 21minutes, 20 minutes or less, e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute.

In another embodiment, the protein-containing acidic solution is thenneutralized by transferring it to a basic solution, for example, bydiafiltering against a basic solution (e.g., a solution of approximately10 mM citrate at a pH of about 5 to 7).

In another embodiment, the protein-containing basic solution istransferred to a final buffer solution, for example, by diafilteringagainst a final buffer solution (e.g., a solution of approximately0.2-0.9% saline (NaCl)).

In another embodiment, the protein-containing final buffer solution canbe further purified, for example, by filtering the solution, forexample, filtering it through a filter having a pore size ofapproximately 0.22 μm.

In another embodiment, the method of the invention further includes thestep of conjugating the purified protein (e.g., antibody) to anadjuvant, such as Keyhole limpet hemocyanin (KLH). Accordingly, thepresent invention can be used to produce a variety of vaccines. In aparticular embodiment, the invention provides a method for producing anantibody vaccine, particularly an antibody against a tumor cell, such asa B cell for the treatment of lymphoma.

The purification unit is an automated apparatus for purifying a proteinor other cell product from a cell product-containing aqueous medium,comprising at least one pre-sanitized or pre-sterilized, disposablecultureware module attached to an automated instrumentation base devicefor controlling liquid flow through the cultureware module. For example,the pre-sanitized or pre-sterilized, disposable cultureware moduleincludes a selection device; multiple, liquid reservoirs; a device forflowing liquid from the reservoirs and into the selection device; adevice for diverting the effluent from the selection device; and adevice for collecting effluent from the selection device. Thepre-sanitized or pre-sterilized, disposable cultureware module canfurther include a diafiltration module; a device for flowing liquid fromthe reservoirs and into the diafiltration module; a device for flowingliquid between the selection device and the diafiltration module; adevice for diverting the effluent from the diafiltration module; and adevice such as a container for collecting effluent from thediafiltration module, e.g., at least two disposable reservoirs.

In a particular embodiment, the device for flowing liquid (such as, washbuffer, elution buffer, or neutralization solution) into the selectiondevice or diafiltration module includes a series of pre-sanitized orpre-sterilized, disposable valves and tubing which connect thereservoirs to the selection device or diafiltration module and whichallow liquid from only one reservoir at a time to pass through theselection device or diafiltration module. Alternatively, the valves andtubing which connect the reservoirs to the selection device anddiafiltration module allow liquid from more than one reservoir at a timeto pass through the selection device.

In one embodiment, the valve includes a disposable outer body throughwhich flexible tubing is threaded. A cammed shaft is mated with the bodyand a motor drives the shaft to open and close the tubing. Multipletubing lines can be controlled by one motor/shaft. The pre-sanitized orpre-sterilized tubing lines contain the fluid and maintain sterility.The tubing and outer body housing are disposed of at the end of use.

In another embodiment, the purification unit includes a device formonitoring the effluent from the selection device or diafiltrationmodule, such as a probe or sensor for measuring the pH, absorbance at aparticular wavelength, or conductivity of the effluent. One or morepressure sensors may be included for monitoring fluid pressure forexcessive pressures, or for control of peristaltic pump speed, e.g., tomaintain the pump speed at a desired pressure (a feedback mechanism). Insome embodiments, the pressure sensor is placed in the purification flowpath, on the output of the pumps.

The pre-sanitized or pre-sterilized, disposable selection device ischosen according to the particular type of purification method used,such as immuno-affinity chromatography, affinity chromatography, ionicexchange chromatography (e.g, anion or cation), hydrophobic interactionchromatography, or size exclusion chromatography (SEC). Suitableselection devices for these types of purification processes are wellknown in the art including, for example, an affinity column packed withan anti-IgM resin, a Protein A, a Protein G, or an anti-IgG resin, anion exchange column containing a charged particle (matrix) which bindsreversibly to particular proteins (e.g., a Vydac VHP-Series ProteinIon-Exchange column with a polystyrene-divinylbenzene copolymer bead anda chemically attached hydrophilic surface), a column packed with ahydrophobic absorbent, such as cellulose, cross-linked dextrose(Sephadex), or a column containing cross-linked polystyrene with poresof varying sizes. The selection device can include a combination ofpurification columns. For example, an affinity chromatography column canbe used, followed by an SEC column to remove any unwanted aggregate.Furthermore, ion exchange chromatography can be used as a “polishingstep” to capture and remove contaminants following affinitychromatography.

In those embodiments in which the selection device is a chromatographydevice, any chromatrography media having surface chemistries capable ofcapturing the cell product may be used. Traditional chromatographymethods use columns packed with porous particles, which may be used inthe invention; however, the architecture of the chromatography is notcritical. For example, the chromatography media may be a membrane,monolith, or porous particles.

In some embodiments, the selection device is a chromatography column orfilter having a natural or synthetic hydroxyapatite matrix (e.g.,ceramic hydroxyapatite). Hydroxyapatite is a naturally occurring mineralform of calcium apatite. Hydroxyapatite is the hydroxyl end member ofthe complex apatite group. The OH— ion can be replaced by fluoride,chloride or carbonate. It crystallizes in the hexagonal crystal system.Hydroxyapatite can be used in chromatography for purification. Themechanism of hydroxyapatite chromatography is somewhat complicated andhas been described as “mixed-mode” ion exchange. It involves nonspecificinteractions between positively charged calcium ions and negativelycharged phosphate ions on the stationary phase hydroxyapatite resin withprotein negatively charged carboxyl groups and positively charged aminogroups. For elution, a buffer with increasing phosphate concentration istypically used. Hydroxyapatite that can be used to pack columns andfilters for chromatography includes natural hydroxyapatite and synthetichydroxyapatite (e.g., crystalline hydroxyapatite, ceramichydroxyapatite). Thus, in some embodiments of the purification method ofthe present invention, solution containing desired immunoglobulin orother cell product can be purified by a hydroxyapatite column or filter,wherein the hydroxyapatite column or filter is packed with naturalhydroxyapatite or synthetic hydroxyapatite. In some embodiments, thesynthetic hydroxyapatite is crystalline hydroxyapatite or ceramichydroxyapatite.

In a particular embodiment, the selection device comprises apre-sterilized affinity purification column, e.g., a columnapproximately 1.5 to 2.5×10 cm in length which is pre-packed withapproximately 4 to 10 ml of resin, such as an affinity ligand (bindingsubstance), such as Protein A, Protein A analogs, Protein G, anti-IgG oranti-IgM resin. Affinity chromatography (AC) is a technique enablingpurification of a biomolecule with respect to biological function orindividual chemical structure. The substance to be purified isspecifically and reversibly adsorbed to a ligand which is immobilized bya covalent bond to a chromatographic bed material (matrix). Samples areapplied under favorable conditions for their specific binding to theligand. Substances of interest are consequently bound to the ligandwhile unbound substances are washed away. Recovery of molecules ofinterest can be achieved by changing experimental conditions to favordesorption.

The ligand, Protein A, is a group specific ligand which binds to the Fcregion of most IgG. It is synthesized by some strains of staphylococcusaureus and can be isolated from culture supernatants then insolubilisedby coupling to agarose beads or silica. An alternative method is to usewhole bacteria of a strain which carries large amounts of Protein A onthe bacterial cell surface. Both types of gel preparation are availablecommercially (Pharmacia; Calbiochem). Alternatively, a recombinant formof Protein-A can be used (ProSep-rA, Millipore).

An alternative to Protein A is Protein G (Anal. Chem. (1989)61(13):1317). Protein G is a cell surface-associated protein fromstreptococcus that binds to IgG with high affinity. It has three highlyhomologous IgG-binding domains.

Anti-IgM antibody can also be used as part of the selection device ofthe present invention to purify antibodies. In a particular embodiment,the anti-IgM antibody includes a mouse anti-human IgM monoclonalantibody attached to sepharose by cyanogen bromide (CNBr).

In one embodiment, the ligand, e.g., the affinity resin, is immobilizedon a solid phase. The solid phase may be a purification column or adiscontinuous phase of discrete particles. In a particular embodiment,the solid phase is a controlled pore glass column or a silicic acidcolumn. Optionally, the solid phase is coated with a reagent (such asglycerol) which prevents nonspecific adherence of contaminants to thesolid phase.

Proteins which can be purified by the present invention include variousforms of proteins, such as tumor antigens and antibodies. An epitope ofthe tumor antigen can be any site on the antigen that is reactive withan antibody or T cell receptor. Other examples of tumor antigensinclude, but are not limited to human epithelial cell mucin (Muc-1; a 20amino acid core repeat for Muc-1 glycoprotein, present on breast cancercells and pancreatic cancer cells), the Ha-ras oncogene product, p53,carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3,GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100,HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA),alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogeneproduct, HPV E7 and melanoma gangliosides, as well as any other tumorantigens now known or identified in the future. In some embodiments, thetumor antigen is the idiotype of a B-cell derived lymphoma (e.g., IgM orIgG isotype).

Various antibody isotypes are also encompassed by the invention,including IgG, IgM, IgA, IgD, and IgE. The antibodies of this inventioncan be isolated from a number of sources, including without limitation,serum of immunized animals, ascites fluid, hybridoma or myelomasupernatants, conditioned media derived from culturing a recombinantcell line that expresses the immunoglobulin molecule and from all cellextracts of immunoglobulin producing cells.

A purified protein, e.g., antibody, of the present invention issubstantially free from host cell contaminants such as host cellproteins, nucleic acids and endotoxins.

In a particular embodiment of the invention, the automated methodinvolves purifying a protein using the steps outlined in Example 1 andthe purification unit as represented schematically in FIG. 32.Specifically, prior to loading the protein-containing aqueous medium,the selection device, e.g., an affinity column of approximately 1.5 to2.5.×10 cm in length packed with approximately 4 to 10 ml of resin, iswashed. The selection device can be washed with liquid stored in theliquid reservoirs which are connected to the selection device withpre-sanitized valves and tubing. For example, the selection device canbe washed with phosphate buffer saline (PBS) or a neutral buffer at a pHof about 7.2 to remove impurities, such as preservatives found in thepre-packed, pre-sterilized, disposable column. The size of the columnmay vary based on the type of protein being purified. For example,methods for purifying IgM antibodies use a column about 2.5 cm indiameter, while methods for purifying IgG antibodies use a column about1.5 cm in diameter.

Optionally, if the cell product to be purified is a membrane-associatedprotein, such as a membrane-bound receptor, the method can furthercomprise freeing the membrane-associated cell product from the membranematerial using, for example, proteolytic enzymes, prior to loading theculture medium containing the cell products onto the selection device.

In addition, before loading, the column can be pre-eluted with anelution buffer stored in the liquid reservoirs, such as a buffer at alow pH of about 2.4 to 3.0. The column can then be equilibrated toneutralize or increase the pH using, e.g., PBS, and is ready forloading.

The protein-containing aqueous medium or supernatant is then loaded ontothe pre-sterilized, disposable selection device. This can be done afteradjusting the supernatant or, more preferably, is done without adjustingthe supernatant. The appropriate rate for loading can be determined asis known in the art and generally involves loading at a rate of at least0.5 to 2.5 ml/min, preferably about 5.0 ml/min.

In a particular embodiment, the medium is heated and/or degassed priorto loading to reduce or eliminate the amount of dissolved gas which canaccumulate in the separation device and hinder its ability to bind cellproduct (e.g., protein). For example, the protein-containing aqueousmedium is heated to about room temperature and degassed.

Once loaded, the selection device can be washed with a wash solutionthat is stored in a liquid reservoir to remove any residual contaminantscontained in the aqueous medium, such as residual proteins from the hostcells which were used to produce the protein to be purified, e.g.,contaminants such as host cell proteins, nucleic acids and endotoxins.The appropriate volume and solution for removing contaminants can bedetermined as is known in the art. In a particular embodiment, thecolumn is washed using a buffer, such as PBS, until the ultraviolet (UV)absorbance of the effluent is about zero as measured using standardphotometric procedures.

The protein is then eluted, for example, by using an acidic solution,thereby producing a protein-containing acidic eluate. In anotherembodiment, the salt concentration of the loaded column is changed. Toelute by changing the pH of the loaded column, an acidic elution buffercan be added, such as an elution buffer containing approximately 0.05 to0.5 M of an acid and at a pH of about 2.0 to 5.0. The appropriate volumeand rate of the elution buffer can be determined by one of ordinaryskill in the art. In a particular embodiment, the elution buffer isadded to the column at approximately 1.0 to 2.0 ml/min for a total ofabout four (4) column volumes. Further, the type of elution bufferdepends on the type of protein to be purified and can also be determinedbased on the techniques known in the art. For example, for purificationof an IgM antibody, the elution buffer may comprise approximately 0.1 Mglycine at about pH 2.4. For purification of an IgG antibody, theelution buffer may comprise approximately 0.1 M citrate at approximatelypH 3.0. Those of ordinary skill in the art can determine the optimummolarity and pH based on the ranges and teachings provided herein.

In a particular embodiment, the elution of the purified protein from theselection device can be aided by a monitoring device. For example, theabsorbance at a particular wavelength of the eluate can be monitoredusing a photometer to determine the appropriate concentration of theeluate. Methods for eluting proteins by using a photometer are wellknown in the art. Generally, collection of the peaks containing thepurified protein begins when the ultraviolet (UV) absorbance of theeluate begins to increase from baseline (zero). Collection continuesuntil the UV absorbance returns to its baseline. In a particularembodiment, the volume of the peak fractions collected is about 10 to 25ml and the peaks are collected in a pre-sanitized or pre-sterilized,reservoir contained within the purification apparatus.

The purification unit and method of the invention can further include aviral clearance step to remove virus from the culture medium containingthe cell product (e.g., by filtration).

Following elution, the purified biological product (e.g., protein suchas antibody) can be collected in a pre-sterilized, disposable collectionvessel and removed from the purification unit. In another embodiment,the eluted biological product (e.g., antibody) can undergo furtherprocessing by the automated purification unit. For example, the elutedbiological product can be transferred to an acidic solution, e.g., anacidic solution containing approximately 0.1 M glycine at a pH ofapproximately 2.4, to ensure that the previous buffer that thebiological product was solubilized in has been replaced by the acidicsolution. Enough volume is used so that the buffer exchange efficiencyis theoretically greater than or equal to about 99.5%. Once in theacidic solution (e.g., 0.1 M glycine at pH 2.4), the protein-containingsolution is treated (held) for less than approximately 16 hours at theseconditions in order to inactivate any susceptible virus that may bepresent. However, holding the protein-containing solution for longerthan 16 hours may result in degradation of the protein. Proteindegradation is caused directly by the low pH which unfolds the proteinirreversibly or by proteases which are more active at low pH.Degradation of proteins can be measured by using assays whichcharacterize the structure of the proteins, such as gel electrophoresisand high performance liquid chromatography (HPLC). Degradation ofproteins can also be measured by protein activity, such as potency,toxicity, or content, in a biological assay, such as in vitro cellreceptor binding assays or in vitro antigen content assays. Accordingly,in one embodiment, the protein is held in the acidic solution forapproximately 15.5 hours, 15 hours, 14.5 hours, 14 hours, 13.5 hours, 13hours, 12.5 hours, 12 hours, 11.5 hours, 11 hours, 10.5 hours, 10 hours,9.5 hours, 9 hours, 8.5 hours, 8 hours, 7.5 hours, 7 hours, 6.5 hours, 6hours, 5.5 hours, 5 hours, 4.5 hours, 4, hours, 3.5 hours, 3 hours, 2.5hours, 2 hours, 1.5 hours, 1 hour or less. In another embodiment, theprotein is held in the acidic solution for less than 1 hour, for examplefor approximately 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25minutes, 24 minutes, 23 minutes, 22 minutes, 21 minutes, 20 minutes orless, e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 minute.

The protein-containing acidic solution can further be neutralized bytransferring it to a neutral or basic solution to neutralize the effectsof the previous low pH treatment, for example, a solution containingapproximately 10 mM citrate at a pH of about 5.3. During this step, acontinual and gradual rise in pH occurs over the course of less thanapproximately 16 hours, e.g., approximately 30 minutes. In a particularembodiment, neutralization is completed within about 29-30 minutes,preferably within about 28-29 minutes, and more preferably within about25-28 minutes of transferring the protein from the acidic solution.

In another embodiment, the purified biological product (e.g., protein,such as antibody) is transferred into an appropriate buffer, such as asaline buffer having approximately 0.2-0.9% saline (NaCl). In aparticular embodiment, enough volume is used so that the buffer exchangeefficiency is theoretically greater than or equal to about 99.5%.

In an additional embodiment, the biological product (e.g., protein, suchas antibody) contained in the final buffer solution is filtered througha filter, for example, a filter having a pore size of approximately 0.22μm. The purified protein is automatically deposited into apre-sterilized collection vessel and removed from the automatedpurification apparatus. In a particular embodiment, the purifiedbiological product (e.g., protein, such as antibody) is stored in asolution containing approximately 0.2-0.9% saline or further processed.

In another embodiment, the step of transferring the eluted antibody todifferent solutions occurs automatically using a pre-sterilizeddiafiltration module. Diafiltration is the fractionation process thatwashes smaller molecules through a membrane and keeps molecules ofinterest in the retentate. Diafiltration can be used to remove salts orexchange buffers. In discontinuous diafiltration, the solution isconcentrated, and the lost volume is replaced by new buffer.Concentrating a sample to half its volume and adding new buffer fourtimes can remove over 96% of the salt. In continuous diafiltration, thesample volume is maintained by the inflow of new buffer while the saltand old buffer are removed. Greater than 99% of the salt can be removedby adding up to seven volumes of new buffer during continuousdiafiltration. In a particular embodiment, the diafiltration modulecontains a filtration membrane of approximately 50 cm² areas having anormal molecular weight limit or cutoff of 50,000 daltons. Specifically,the diafiltration module is used to further purify the protein (e.g.,the antibody) and uses the tangential flow filtration principle wherebymolecules over 50,000 daltons (e.g., the antibodies, such as IgG andIgM) cannot pass through the membrane but small molecules, such asbuffers, can pass through. Accordingly, the diafiltration module can beused to exchange one buffer for another and is a more efficientsubstitute for dialysis. Diafiltration can be used to neutralize pH andas a concentration step (to concentrate the cell product).

In a particular embodiment, the diafiltration module is sanitized usinga solution containing approximately 0.1 N Sodium Hydroxide at acrossflow or feed rate of approximately 20-40 mL/min. This crossflowrate is maintained throughout the process. The 0.1 N Sodium Hydroxide isflushed out of the system using a solution containing approximately 0.1M Glycine at a pH of about 2.4. After sanitization is complete, theprotein-containing solution which was eluted from the selection deviceis introduced into the diafiltration module.

In a particular embodiment, the present invention provides an automatedmethod of producing a vaccine by purifying a protein and conjugating theprotein to an adjuvant. More particularly, the invention provides amethod for producing an autologous vaccine, i.e., a vaccine, such as anantibody vaccine, against a self-protein or idiotype (ID) antigen, suchas a tumor antigen. In a particular embodiment, the antigen is a B cellantigen, such as an antibody expressed on B cell tumors (e.g.,lymphomas). Accordingly, the vaccine is used to target one specificmolecule which is expressed by B-cell lymphoma cells. Moreover, sinceeach vaccine produced is patient-specific, the one time, disposable useof the cultureware used in the invention is particularly advantageous.

Examples of adjuvants include, for example, keyhole limpet hemocyanin(KLH), bovine serum albumin, (BSA), and β₂-glycoprotein I. Otheralbumins such as ovalbumin, mouse serum albumin or rabbit serum albumincan also be used as adjuvants, as well as bovine gamma globulin ordiphtheria toxoid.

KLH is a respiratory protein found in mollusks. Its large size (M.W.8-9×10⁶ Da) makes it very immunogenic and the large number of lysineresidues available for conjugation make it very useful as a carrier forhaptens. The phylogenic separation between mammals and mollusksincreases the immunogenicity and reduces the risk of cross-reactivitybetween antibodies against the KLH carrier and naturally occurringproteins in mammalian samples. KLH is obtainable both in its nativeform, for conjugation via amines, and succinylated, for conjugation viacarboxyl groups. Succinylated KLH may be conjugated to a haptencontaining amine groups (such as a peptide) via cross-linking withcarbodiimide between the newly introduced carboxyl groups of KLH and theamine groups of the hapten. Protocols for conjugating haptens to carrierproteins may be found in Antibodies: A Laboratory Manual, E. Harlow andD. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y.,1988) pp. 78-87.

Accordingly, in one embodiment, the invention provides an automatedmethod of producing a vaccine by purifying an antibody and conjugatingthe antibody to KLH. Methods for conjugating proteins (e.g., antibodies)to adjuvants (e.g., KLH) are known in the art. In general, conjugationis achieved by mixing the purified protein and the adjuvant with anappropriate catalyst under the appropriate conditions. In a particularembodiment, it may be necessary to take a sample of the purified protein(e.g., antibody) for off-line determination of the antibodyconcentration before the adjuvant (e.g., KLH) is added. In anotherparticular embodiment, gluteraldehyde is added to begin the conjugation.Since gluteraldehyde is unstable, it may be added manually and can beadded in concentrated form or a more dilute form. Adding gluteraldehydein dilute form may require a diafiltration or dialysis step. Afterseveral hours, the conjugation is quenched. In a particular embodiment,the conjugation is quenched by adding glycine. The conjugated protein(e.g., antibody) can be purified further using techniques known in theart, such as dialysis. In a particular embodiment, the conjugatedprotein is dialyzed using saline for injection (SFI) and the product isrecovered.

The purification method and apparatus of the present invention can beused in conjunction to provide a fully automated method for purifyingproteins.

Conventionally, each unique cell line must be cultured, cell secretionsharvested and purified separately. In order to manage a large number ofunique cell lines, as for example might be required for the productionof large numbers of autologous cell therapeutic products or largenumbers of unique monoclonal antibodies, a considerable number ofinstruments would be needed. Compactness of the design and the amount ofancillary support resources needed become an important facilities issue.Small stirred tank systems require a device of steam generation anddistribution (for steam-in-place sterilization) or autoclaves tosterilize the vessels and supporting plumbing. To support a large numberof units becomes a logistics problem for the facility. The apparatus ofthe present invention has no such requirement. Larger scale cell cultureis historically done in segregated steps that often require separatetypes of equipment. Manual handling, storage and tracking is needed forall these steps as the culture expands and product is harvested. Themethod of the present invention integrates these steps into acontinuous, fully integrated sequential process. This eliminates thehandling risk and facilitates the data gathering required for thoroughdocumentation of the entire process.

Exemplified Embodiments

Embodiment 1: An automated cell culture and purification apparatus forthe production of cells and cell derived products, comprising:

(a) a cell culture unit comprising:

a first reusable instrumentation base device incorporating hardware tosupport cell culture growth; and

at least one first disposable cell cultureware module removablyattachable to said first instrumentation base device, said firstcultureware module including a cell growth chamber; and

(b) a purification unit linked to said cell culture unit, saidpurification unit comprising:

a second reusable instrumentation base device incorporating hardware toreceive fluid from said cell growth chamber of said cell culture unit;and

at least one second disposable cell cultureware module removablyattachable to said second instrumentation base device of saidpurification unit, said second cultureware module including a selectiondevice (such as a purification column).

Embodiment 2: The cell culture and purification apparatus of embodiment1, wherein said first instrumentation device of said cell culture unitincludes a pump for circulating cell culture medium through the at leastone cultureware module.

Embodiment 3: The cell culture and purification apparatus of embodiment2, wherein the pump of said cell culture unit moves growth factor orother supplements into the cell growth chamber and removes productharvest from the cell growth chamber.

Embodiment 4: The cell culture and purification apparatus of embodiment2, wherein said first instrumentation device of said cell culture unitincludes a plurality of rotary selection valves to control the mediumflow through the at least one first cultureware module of said cellculture unit.Embodiment 5: The cell culture and purification apparatus of embodiment1, wherein said first instrumentation device of said cell culture unitincludes a cool storage area for storing growth factor or othersupplements and product harvest.Embodiment 6: The cell culture and purification apparatus of embodiment1, wherein said first instrumentation device of said cell culture unitincludes a heating mechanism for heating the cell growth chamber topromote growth and production.Embodiment 7: The cell culture and purification apparatus of embodiment6, wherein said at least one first cultureware module of said cellculture unit includes an inlet and outlet port, said inlet and outletports being constructed and arranged to align with air ports of saidinstrument device of said cell culture unit such that the heat exchangemechanism forces heated air into said at least one first culturewaremodule from said first instrument device of said cell culture unit.Embodiment 8: The cell culture and purification apparatus of embodiment2, further comprising a pump cassette having attached tubing, the pumpcassette and tubing being insertable into the multi-channel pump.Embodiment 9: The cell culture and purification apparatus of embodiment2, wherein said at least one first cultureware module includes a gasblending mechanism in communication with the cell growth chamber.Embodiment 10: The cell culture and purification apparatus of embodiment9, further comprising a pH sensor disposed in said at least one firstcultureware module to control the pH of the cell culture medium.Embodiment 11: The cell culture and purification apparatus of embodiment10, wherein the gas blending mechanism includes a gas exchange cartridgethat provides oxygen and adds or removes carbon dioxide to the medium tosupport cell metabolism.Embodiment 12: The cell culture and purification apparatus of embodiment11, wherein the gas exchange cartridge has an inlet end and a dischargeend.Embodiment 13: The cell culture and purification apparatus of embodiment12, further comprising a carbon dioxide sensor in fluid communicationwith the discharge end of the gas exchange cartridge for measuring thecarbon dioxide level of the cell culture medium.Embodiment 14: The cell culture and purification apparatus of embodiment1, wherein said at least one first cultureware module of said cellculture unit is pre-sterilized, and wherein said at least one secondcultureware module of said purification unit is pre-sterilized.Embodiment 15: The cell culture and purification apparatus of embodiment1, wherein said at least one first cultureware module of said cellculture module includes a plurality of interface features integratedinto said first cultureware module that mate with instrument interfacefeatures in said first instrumentation device.Embodiment 16: The cell culture and purification apparatus of embodiment2, wherein said at least one first cultureware module of said cellculture unit includes sensors for sensing fluid circulation rate,temperature and pH of the cell culture medium.Embodiment 17: The cell culture and purification apparatus of embodiment1, wherein the cell growth chamber comprises a bioreactor that providescell space and medium component exchange.Embodiment 18: The cell culture and purification apparatus of embodiment1, wherein the selection device is selected from the group consisting ofaffinity chromatography, immuno-affinity chromatography, ionic exchangechromatography, hydrophobic interaction chromatography, and sizeselection chromatography (SEC), or a combination of two or more of theforegoing chromatography devices.Embodiment 19: The cell culture and purification apparatus of embodiment1, wherein said at least one first cultureware module includes a fluidcycling unit disposed therein to cycle and maintain fluid volumes withinthe cell growth chamber.Embodiment 20: The cell culture and purification apparatus of embodiment19, wherein the fluid cycling unit includes a non-rigid reservoir and asecond flexible reservoir in fluid communication with the firstreservoir to cause elevated pressure in the first reservoir.Embodiment 21: The cell culture and purification apparatus of embodiment1, further comprising a plurality of disposable containers for harvestcollection and flushing removably connected to said at least one firstcultureware module.Embodiment 22: The cell culture and purification apparatus of embodiment1, wherein said selection device comprises an affinity resin.Embodiment 23: The cell culture and purification apparatus of embodiment22, wherein said affinity resin is selected from the group consisting ofanti-IgM resin, Protein A, Protein G, and anti-IgG resin.Embodiment 24: The cell culture and purification apparatus of embodiment1, wherein said selection device comprises a chromatography devicecomprising hydroxyapatite resin.Embodiment 25: The cell culture and purification apparatus of embodiment1, wherein said second disposable cultureware module further comprises:multiple, liquid reservoirs, device for flowing liquid from thereservoirs and into the selection device, device for diverting theeffluent form the selection device, and device for collecting effluentfrom the selection device.Embodiment 26: The cell culture and purification apparatus of embodiment25, wherein the device for flowing liquid into the selection devicecomprises a series of pre-sterilized, disposable valves and tubing whichconnect the reservoirs to the selection device and which allow liquidfrom only one reservoir at a time to pass through the selection device.Embodiment 27: The cell culture and purification apparatus of embodiment25 or 26, wherein the device for flowing liquid into the selectiondevice comprises a series of pre-sterilized, disposable valves andtubing which connect the reservoirs to the selection device and whichallow liquid from more than one reservoir at a time to pass through theselection device.Embodiment 28: The cell culture and purification apparatus of embodiment1, wherein said second cultureware module of said purification unitfurther comprises a device for diafiltering the purified product.Embodiment 29: The cell culture and purification apparatus of embodiment1, wherein said purification unit further comprises a device formonitoring the effluent from the selection device.Embodiment 30: The cell culture and purification apparatus of embodiment29, wherein the device for monitoring involves measuring the pH,absorbance at a particular wavelength, or conductivity of the effluent.Embodiment 31: The cell culture and purification apparatus of embodiment25, wherein the multiple liquid reservoirs each contain a wash buffer,an elution buffer, or a neutralization solution.Embodiment 32: The cell culture and purification apparatus of embodiment31, wherein the multiple liquid reservoir comprises two reservoirs, andwherein each of the two reservoirs contains a wash buffer.Embodiment 33: The cell culture and purification apparatus of embodiment25, wherein the pre-sterilized, disposable device for collectingeffluent from the selection device includes at least two disposablereservoirs.Embodiment 34: An automated method for the production of cells and cellproducts and purification thereof in a contaminant-free environment,comprising the steps of:

providing at least one first disposable cultureware module, said firstmodule including a cell growth chamber;

providing a first reusable instrumentation base device incorporatinghardware to support cell culture growth, said base device including amicroprocessor control and a pump for circulating cell culture mediumthrough the cell growth chamber;

providing at least one second disposable cultureware module, said secondcultureware module including a selection device (such as a purificationcolumn);

providing a second reusable instrumentation base device incorporatinghardware to receive fluid from the cell growth chamber;

removably attaching said at least one first cultureware module to saidfirst instrumentation base device;

introducing cells into the cell growth chamber;

fluidly attaching a source of cell culture medium to said at least onefirst cultureware module;

programming operating parameters into the microprocessor control;

operating the pump to circulate the cell culture medium through the cellgrowth chamber to grow cells or cell products therein;

loading the culture medium containing the cell products onto theselection device to absorb a cell product onto the selection device;

eluting the absorbed cell product into an aqueous medium; and

collecting the cell product in a pre-sterilized disposable collectionvessel to form a purified cell product.

Embodiment 35: The method of embodiment 34, wherein the selection deviceis selected from the group consisting of affinity chromatography,immuno-affinity chromatography, ionic exchange chromatography,hydrophobic interaction chromatography, and size selectionchromatography (SEC), or a combination of two or more of the foregoing.Embodiment 36: The method of embodiment 34, wherein the selection devicecomprises a chromatography device comprising hydroxyapatite resin.Embodiment 37: The method of embodiment 34, further comprising disposingof said at least one first cultureware module and said at least onesecond cultureware module.Embodiment 38: The method of embodiment 34, wherein said at least onefirst cultureware module includes a gas exchange unit and furthercomprising the step of providing oxygen and adding or removing carbondioxide to the cell culture medium to support cell metabolism.Embodiment 39: The method of embodiment 34, wherein said at least onefirst cultureware module includes a pH sensor disposed therein andfurther comprising the step of controlling the pH of the cell culturemedium.Embodiment 40: The method of embodiment 38, further comprising the stepof regulating the cell culture medium feed rate control of the medium.Embodiment 41: The method of embodiment 40, wherein the step ofregulating the cell culture medium feed rate control includes monitoringcarbon dioxide levels in the cell growth chamber to calculate lactateconcentration of the cell culture medium.Embodiment 42: The method of embodiment 41, wherein the step ofregulating includes calculating an initial bicarbonate level of the cellculture medium and utilizing the measured pH and carbon dioxide level ofthe cell culture medium to calculate the lactate concentration.Embodiment 43: The method of embodiment 34, further comprising the stepof heating the at least one first cultureware module to promote cellgrowth.Embodiment 44: The method of embodiment 34, further comprising the stepof pumping high molecular weight factor into the cell growth chamber.Embodiment 45: The method of embodiment 44, wherein said firstinstrumentation base device includes a cool storage area, and saidmethod further comprises the step of storing the high molecular weightfactor and product harvest in the cool storage area.Embodiment 46: The method of embodiment 34, wherein said culturewaremodule has an identifying code and further comprising the step ofscanning the identifying code information into the microprocessorcontrol.Embodiment 47: The method of embodiment 34, further comprising the stepof pre-sterilizing said at least one cultureware module.Embodiment 48: The method of embodiment 34, wherein said at least onecultureware module includes a plurality of interface features integratedinto the module and said step of attaching said at least one culturewaremodule to said instrumentation base device includes mating the moduleinterface features with interface features on said instrumentation basedevice.Embodiment 49: The method of embodiment 34, wherein said at least onecultureware module includes a plurality of sensors and furthercomprising the step of sensing fluid circulation rate, temperature andpH of the cell culture medium.Embodiment 50: The method of embodiment 34, wherein said at least onecultureware module includes a fluid cycling unit disposed therein andfurther comprising the step of cycling and mixing fluid of the cellculture medium within the cell growth chamber.Embodiment 51: The method of embodiment 50, wherein cycling is achievedby utilizing a sealed flexible reservoir for the EC reservoir and thestep of cycling comprises cycling the cell culture medium in and out ofthe flexible reservoir.Embodiment 52: The method of embodiment 51, wherein the step of cyclingfurther comprises using a second flexible reservoir is used to applyindirect pressure to the EC reservoir to effect cycling of the cellculture medium.Embodiment 53: The method of embodiment 34, further comprising the stepof attaching another disposable cultureware module after the step ofdisposing of said at least one cultureware module.Embodiment 54: The method of embodiment 34, further comprising the stepof directing an operator through a sequenced run of the cell cultureprocess with the microprocessor control.Embodiment 55: The method of embodiment 34, further comprising the stepof transferring the product into an acidic solution.Embodiment 56: The method of embodiment 55, wherein the transfer isachieved by diafiltration.Embodiment 57: The method of embodiment 55, wherein the product is heldin the acidic solution.Embodiment 58: The method of embodiment 34, further comprising the stepof transferring the product (antibody) to a basic solution.Embodiment 59: The method of embodiment 58, wherein transfer is achievedby diafiltration.Embodiment 60: The method of embodiment 59, further comprising the stepof transferring the product to a final buffer solution.Embodiment 61: The method of embodiment 60, wherein the transfer isachieved by diafiltration.Embodiment 62: The method of embodiment 34, further comprising the stepof washing the selection device with at least one solution prior toloading the medium.Embodiment 63: The method of embodiment 34, further comprising the stepof washing the selection device with at least one solution prior toeluting the product.Embodiment 64: The method of embodiment 63, wherein the at least onesolution comprises the use of PBS, glycine, and/or citrate.Embodiment 65: The method of embodiment 34, further comprising the stepof warming and/or degassing the medium prior to loading the medium ontothe selection device.Embodiment 66: The method of embodiment 34, wherein the step of elutingthe product comprises the use of a solution containing glycine orcitrate.Embodiment 67: The method of embodiment 66, wherein the solutioncomprises approximately 0.1 M glycine at a pH of about 2.4 orapproximately 0.1 M citrate at a pH of about 3.0.Embodiment 68: The method of embodiment 34, wherein the step of elutingthe product comprises the use of a photometer.Embodiment 69: The method of embodiment 55, wherein the step oftransferring the product into an acidic solution comprises the use of asolution containing glycine.Embodiment 70: The method of embodiment 69, wherein the solutioncomprises approximately 0.1 M glycine at a pH of about 2.4.Embodiment 71: The method of embodiment 58, wherein the step oftransferring the product to a basic solution comprises the use of asolution containing citrate.Embodiment 72: The method of embodiment 69, wherein the basic solutioncomprises approximately 10 mM citrate at a pH of about 5.3.Embodiment 73: The method of embodiment 60, wherein the step oftransferring the product to a final buffer solution comprises the use ofsolution containing saline.Embodiment 74: The method of embodiment 73, wherein the final buffersolution comprises approximately 0.2-0.9% saline.Embodiment 75: The method of embodiment 60, further comprising the stepof filtering the product contained in the final buffer solution prior tocollecting the product in a pre-sterilized, disposable collectionvessel.Embodiment 76: The method of embodiment 34, wherein the selection devicecomprises a column packed with an affinity resin.Embodiment 77: The method of embodiment 76, wherein the affinity resinis anti-IgM resin.Embodiment 78: The method of embodiment 76, wherein the affinity resinis selected from the group consisting of a Protein A, Protein G, andanti-IgG resin.Embodiment 79: The method of embodiment 34, wherein the product is anantibody.Embodiment 80: The method of embodiment 79, wherein the antibodycomprises IgM, IgG, IgD, IgE, or IgA.Embodiment 81: The method of embodiment 79, wherein the antibodycomprises IgM.Embodiment 82: The method of embodiment 79, wherein the antibody targetsan idiotype antigen expressed on B cell tumors.Embodiment 83: An automated method for producing an immunogeniccomposition such as a vaccine, comprising:

purifying an antibody from an antibody-containing aqueous medium usingthe method according to any of embodiments 34-82; and

conjugating the purified antibody to an adjuvant.

Embodiment 84: The method of embodiment 83, wherein the adjuvantcomprises keyhole limpet hemocyanin (KLH).

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

All patents, patent applications, provisional applications, andpublications referred to or cited herein, supra or infra, areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Automated Purification Technique for Immunoglobulins

In a particular embodiment of the invention, the automated methodinvolves purifying an antibody (e.g., IgM or IgG) using the stepsoutlined below and using the purification unit shown schematically inFIG. 32.

Supernatant Source: Filtered supernatant containing the unpurifiedantibody (IgM, IgG, or other immunoglobulin isotype) is produced in anautomated cell culture device (e.g., the AUTOVAXID™ cell culturemodule). Once the desired quantity of antibody has been produced (e.g.,approximately 100 mg of antibody) the automated purification module isconnected to the cell culture device. The filtered supernatant istransferred to the automated purification unit by a pump.

Column Set-Up: For IgM, a pre-sterilized, disposable, glass 2.5×10 cmcolumn is packed with anti-IgM chromatography resin and snapped into theautomated unit which also includes a UV monitor and electronic datarecorder. Using the automated purification unit as shown in FIG. 32, PBSis flowed through the column at a pump rate of 5 mL/min. This same pumprate is used throughout the process. The column is pre-eluted with 0.1 Mglycine at pH 2.4. The column is then equlibrated with PBS.

For IgG, a pre-sterilized, disposable 1.5×10 cm column is packed withProtein A chromatography resin and snapped into the automated apparatuswhich also includes a UV monitor and chart recorder. Using the automatedpurification unit as shown in FIG. 32, the system is primed with PBS ata pump rate of 5 mL/min. The column is pre-eluted with 0.1 M citrate ata pH of 3.0. The column is then equlibrated with PBS.

Purification: For IgM, the filtered supernatant is passed through aheater to warm it to ambient temperature prior to entering the column.Once all of the supernatant has entered the column, the column is washedwith PBS to remove unbound impurities. The column is then further washedwith PBS at a pH of 5. The purified IgM is eluted from the column using0.1 M glycine at a pH of 2.4. The UV absorbing peak containing thepurified IgM is collected and sent to a mixing chamber for furtherprocessing.

For IgG, the filtered supernatant is passed through a heater to warm itto ambient temperature prior to entering the column. Once all of thesupernatant has entered the column, the column is washed with PBS toremove unbound impurities. The purified IgM is eluted from the columnusing 0.1 M citrate at a pH of 3.0. The UV absorbing peak containing thepurified IgG is collected and sent to a mixing chamber for furtherprocessing.

Sanitization of Diafiltration Apparatus: A membrane basedultrafiltration device is installed in the automated purificationmodule. This cassette or device contains a membrane of 50 cm² areahaving a normal molecular weight limit or cutoff of 50,000 daltons. Thisdevice works on the tangential flow filtration principle wherebymolecules over 50,000 daltons, such as the IgG and IgM, cannot passthrough the membrane but small molecules, such as buffers can passthrough. The tangential flow filtration cassette is used to exchange onebuffer for another and is a more efficient substitute for dialysis.After installation, the tangential flow filter (TFF) cassette and systemis sanitized using 0.1N Sodium Hydroxide at a crossflow or feed rate of20-40 mL/min. This crossflow rate is maintained throughout the process.After sanitization is complete the 0.1N Sodium Hydroxide is flushed outof the system using 0.1M Glycine at a pH of 2.4. The antibody is thenintroduced into the system.

Low pH Treatment: The remaining steps are the same for either IgM orIgG. The antibody is diafiltered against enough volume of 0.1M GlycinepH 2.4 to ensure that the previous buffer that the antibody wassolubilized in has been replaced by the 0.1M Glycine pH 2.4. Enoughvolume is used so that the buffer exchange efficiency is theoreticallygreater than or equal to 99.5%. Once in 0.1M Glycine pH 2.4, theantibody is treated (held) for 30 minutes at these conditions in orderto inactivate any susceptible virus that may be present.

Neutralization: After the 30 minute treatment the antibody isdiafiltered against 10 mM Citrate at a pH of 5.3. The antibody isdiafiltered against sufficient 10 mM Citrate at a pH of 5.3 toneutralize the effects of the previous low pH treatment.

Final Buffer Exchange: After neutralization, the antibody is diafilteredinto 0.2-0.9% Saline (NaCl). Again, enough volume is used so that thebuffer exchange efficiency is theoretically greater than or equal to99.5%. Once in the final buffer the antibody is filtered through a 0.22μm filter and removed from the automated purification unit. Thisfiltered purified antibody can be stored in 0.2-0.9% Saline or furtherprocessed.

The foregoing particular embodiment is summarized below.

Purification Module

1) Prosep A column used for IgG purification

4 mL Prosep-rA Column 1.5 cm diam 5 mL/min

Sanitization: 0.3% HCl pH 1.5

Pre-Equilibration: PBS pH 7.2

Pre-Elution: 0.1M Citrate pH 3.0

Equilibration: PBS pH 7.2

Load: Undiluted Culture Harvest

Post Load Wash: PBS pH 7.2

Elution: 0.1M Citrate pH 3.0

Discard after one use

2) 1D12-CL4B (anti-IgM antibody) column used for IgM purification

9 mL 1D12-Sepharose 4B Column 2.5 cm diam 5 mL/min

Pre-Equilibrate: PBS pH 7.2

Pre-Elution: 0.1M Glycine pH 2.4

Equilibration: PBS pH 7.2

Load: Undiluted Culture Harvest

Post Load Wash 1: PBS pH 7.2

Wash 2: PBS pH 5

Elution: 0.1M Glycine pH 2.4

Discard after one use

3) Material coming from 1 and 2 enters diafiltration module of thecultureware. Diafiltration using Pellicon XL Biomax 50 screen channel A.This would also include the 30 minute low pH hold to inactivatesusceptible virus.

Pellicon XL Biomax 50 membrane area=50 cm² 20-40 mL/min cross flowrate

Sanitize: 0.1N NaOH

Hold: ≥30 minutes at pH 2.4

Flush: 0.1M Glycine pH 2.4

Fill Mix Chamber with Glycine pH 2.4

Precondition: 0.1M Glycine pH 2.4

Fill Mix Chamber with Ab

Diafilter against: 0.1M Glycine pH 2.4

Hold:

Diafilter Antibody against 10 mM Citrate pH 5.3

Diafilter Antibody against 0.2% to 0.9% Saline

Remove Antibody from system through 0.22 μm Filter 10 mL/min

Discard Pellicon after one use

Example 2 Verification of the IgM Purification Process for the AutomatedDownstream Processing Instrument

The purpose of this experiment was to verify the IgM purificationprocess using the purification unit of the invention. The experimentsuccessfully verified the suitability of the human IgM purificationplatform and verified that the process can achieve satisfactory productquality and process performance by purifying three different IgMmolecules from cell culture supernatant to final product.

IgM was purified from cell culture batches (crude supernatant) using acombination of filtration and chromatography-based unit operations.Briefly, the cell free harvest is loaded onto a ceramic hydroxyapatite(CHT) chromatography column where IgM is bound to the resin via a metalaffinity mode. The bound antibody was released from the resin using astep change in the phosphate concentration of the mobile phase. The CHTprocess step removes a significant amount of process-related impurities.Following this initial purification step, the protein containingsolution was virally inactivated using low pH for greater than 35minutes, followed by neutralization and buffer exchange using 50 KDcassettes (Millipore) operated in tangential filtration mode. Theproduct was then loaded onto a flow through (non-binding) Anion Exchange(AEX) chromatography step using Bio-Rad's UNOspere Q to removenegatively charged impurities such as DNA, and Host Cell Protein (HCP).The product was then aseptically recovered though a sterilizing grade0.2 micron final filter. The human IgM purification process issummarized in FIG. 35.

The results of this study demonstrate that the purification unit canrapidly execute the process steps in a highly automated fashion anddeliver product yield of 25 to 52%, host cell protein clearance of1.8-2.1 logs and expected product quality.

The purification unit is a self-contained device that incorporatessingle-use cultureware to purify monoclonal antibodies and otherbiological molecules. Development and evaluation of the prototypeinstrument confirmed the suitability for a generic human IgMpurification platform and verified that the process will result insatisfactory product quality and process performance. The followingdescribes the verification testing with three human IgMs purified usingthe purification platform.

Materials and Methods

Purification unit. Purification was performed using an automatedpurification unit manufactured by Biovest International, Inc. Theinstrument includes of two parts; a reusable chassis that containspumps, valve actuators, a pressure sensor and a UV spectrophotometer;and a disposable set of “cultureware” that consists of all productcontact surfaces such as tubing, valves, columns, filters, etc. Thecultureware is intended to be used once and then discarded.

Cell Culture Supernatant. Cell culture supernatant was collected from 3separate hollow fiber cell culture runs and stored at −20° C. untilpurification. The material was thawed at 2-8° C. prior to furtherprocessing. The material was retained from previous work performed byBiovest and the batches were identified as P92-0349, P92-0375 andP92-0073.

Buffers and Solutions. Buffers and solutions (USP grade) were preparedby combining acidic and basic components such that additional pHadjustment is reduced. All buffers and solutions were sterile filteredprior to use. Recipes for the buffers and solutions used in the processmay be found below under “Buffer and Solution Preparation Recipes”.

Chromatography. Capture chromatography was performed using CeramicHydroxyapatite Type II chromatography resin (Bio-Rad). A 2.5 cm 8.5 cmcolumn (CV=41.7 mL) was packed in a Sigma-Aldrich column and adaptor(Catalog Number C4669). The column was sanitized for 30 minutes in 0.1MNaOH and then pre-equilibrated in 3 CV of 125 mM Phosphate, 25 mM NaClpH 7.0 and then equilibrated in 10 mM Phosphate, 25 mM NaCl, pH 7.0.After equilibration, the column was loaded with room temperature cellculture supernatant to a maximum capacity of 12 mg/ml. The cell culturesupernatant was mixed 1:2 with 10 mM Phosphate, 25 mM NaCl, pH 7.0during load via in-line dilution. After loading the column was washed in5 CV of 10 mM Phosphate, 500 mM NaCl and then further washed with anadditional 5 CV of 10 mM Phosphate, 25 mM NaCl, pH 7.0. The product waseluted with a step change in phosphate using 125 mM Phosphate, 25 mMNaCl, pH 7.0. The UV was monitored and the eluate was collected from 0.5O.D. rising to 0.5 O.D. descending. All operations of this process stepwere performed at 5 mL/min (55 cm/hr). As product eluted from thecolumn, it was continuously transferred to the tangential flow (TFF)loop for subsequent processing.

Low pH Tangential Flow. Prior to product elution from the CHT column,the TFF loop was sanitized for 60 minutes in 0.1M NaOH, followed byequilibration with 80 L/M² of 20 mM citrate pH 3.5. After product wascollected from the CHT column, a constant 50 mL volume was maintained inthe mix chamber during TFF operations. The diluted pool was thendiafiltered against approximately 3 diavolumes (DV) of 20 mM citrate, pH3.5, to reduce the pH to 3.5+/−0.2. Once the Diafiltration was completethe product was held for at least 35 minutes to inactivate endogenousretroviral-like particles or adventitious viruses. All operations duringthis process step were performed under constant pressure with a maximumcross flow rate of 40 mL/min and a maximum inlet pressure of 20 PSIG.

TFF to Final Buffer. At the end of the 35 minute viral inactivationhold, diafiltration resumed with phosphate buffered saline (PBS), pH7.4. Diafiltration in PBS continued for 9 DV to neutralize the productand ensure complete diafiltration into the buffer required forsubsequent processing and product storage. All operations during thisprocess step were performed under constant pressure with a maximum crossflow rate of 40 mL/min and a maximum inlet pressure of 20 PSIG.

Anion Exchange and Product Recovery. After TFF was complete, the productwas recovered from the TFF loop and processed over a Bio-Rad UNOspere Qanion exchange cartridge. The AEX cartridge was sanitized (at the sametime as the TFF loop) and equilibrated in PBS, pH 7.4. Following AEXcartridge equilibration, the final filter was placed in line andequilibrated. The product was processed across these two steps inseries. These process steps were performed at a maximum constantpressure of 25 PSIG. The product was passed through a 0.2 micron filterand collected aseptically in a 250 mL Stedim flexboy bag. Followingproduct collection, an additional 20 mL of PBS was introduced into theTFF loop and passed through the AEX cartridge and 0.2 micron filter torecover any product held up in the system. This flush was collected inthe same bag with the product.

Analytical. All analytical work in support of this study was performedby Biovest's analytical development group. Yield was determined bycomparing total milligrams of IgM loaded with the total milligramsrecovered. IgM concentration was determined by human IgM ELISA. Impurityclearance was quantified by total log reduction of HCP across the entirepurification process. HCP concentrations are determined by HCP ELISA andproduct quality is illustrated by a Coomassie stained, reduced SD S-PAGEof the load material and final product.

Results and Discussion

Process Performance

The purification unit demonstrated a product recovery of 46.7-51.9%.Product volume ranged from 67.5-71.8 mL. The concentration of the finalproduct was directly proportional to the amount of protein loaded andranged from 0.238-1.09 mg/mL as determined by human IgM ELISA.Processing times ranged from 7.5-11.0 hours start to finish depending onthe initial supernatant volume (Table 1).

TABLE 1 Purification Process Performance Load Prod. Sample # Load LoadIgM Sample # Product Prod. IgM Total IgM Batch RD2010- Vol.Concentration mg RD2010- Vol. Concentration Recovered ID 005- (mL)(mg/mL) loaded 005- (mL) (mg/mL) (mg) Yield % P92- 010 353.5 0.238 84.1007 67.5 0.582 39.3 46.7 0349 P92- 004 785.1 0.166 130.3 008 71.8 0.45032.3 24.7 0375 P92- 005 272.2 1.09 296.5 009 70.3 2.01 141.3 51.9 0073Impurity Clearance

The purification process, as described above, demonstrated the abilityto clear approximately XX Logs of HCP. All of the product pools had lessthan 29,800 ng/ml of HCP (Table 2). HCP clearance appears to correlatewith load volume.

TABLE 2 HCP Clearance across Purification Process Load Product SampleLoad HCP Sample Product HCP Number Concen- Number Concen- RD2010-tration RD2010- tration HCP Log Batch ID 005- (ng/mL) 005- (ng/mL)Clearance P92-0349 010 1670000 007 23000 1.8 P92-0375 004 847000 00829800 1.4 P92-0073 005 829000 009 7310 2.1Product Quality

SDS-PAGE analysis shows that the purification process reduces the levelsof high and low molecular weight impurities such as host cell proteinsand aggregated and fragmented IgM. The product samples primarily displaythe characteristic heavy and light chain bands of reduced IgM. BatchesP92-0375 and P92-0073 displays doublets in the light chain region, butthis pattern is evident in the load material as well. This extra bandmay the J-chain found in pentameric IgM or it is possible that the IgMin these load materials degraded during storage, but this pattern doesnot appear to be an artifact of the purification process. See FIG. 36and Table 3 for details.

TABLE 3 SDS-PAGE Gel Lane Descriptions Lane Description 1 See Blue Plus2 MWM 2 Blank 3 RD2010-002-087 P92-0349 Supernatant 5 μg load 4RD2010-005-004 P92-0375 Supernatant 5 μg load 5 RD2010-005-005 P92-0073Supernatant 5 μg load 6 RD2010-005-007 P92-0349 Product 5 μg load 7RD2010-005-008 P92-0375 Product 5 μg load 8 RD2010-005-009 P92-0073Product 5 μg load 9 IgM Standard (commercial) 5 μg load 10 See Blue Plus2 MWM

The platform human IgM purification process is suitable forimplementation on the purification unit. Using an automated combinationof integrated chromatography and filtration process steps, theinstrument is capable of generating purified human IgM in less thaneight hours. The implemented process demonstrates acceptable yield ofgreater than 25% and impurity clearance of greater than 1.5 Logs acrossa variety of IgM molecules and product loading.

Critical Process Filters and Chromatography Resins

TABLE 4 Process Filters and Chromatography Resins Vendor CatalogResin/Filter Name Purpose Number Bio-Rad Ceramic Metal Affinity Capture157-2000 Hydroxyapatite Type II Chromatography Millipore Biomax 50-50cm² Ultrafiltration/Diafiltration PXB050A50 Bio-Scale Mini UNOspereAnion Exchange 732-4102 Q Cartridge Chromatography Sartolab P20 PlusSterile Product Filtration 18058DBuffer and Solution Preparation Recipes

TABLE 5 0.1M NaOH - Chromatography Column and System SanitizationSolution Component Molarity (mM) g/L Sodium Hydroxide 100 4.0 pH ≥12Conductivity (mS/cm at 24-26° C.) 19-24

TABLE 6 10 mM Phosphate 25 mM NaCl, pH 7.0 - CHT Equilibration and Wash2 Buffer Component Molarity (mM) g/L Sodium Phosphate (monobasic) 4.70.635 Sodium phosphate (dibasic-12 hydrate) 5.3 1.90 Sodium Chloride 251.46 pH 6.8-7.2 Conductivity (mS/cm at 24-26° C.) 3-7

TABLE 7 125 mM Phosphate 25 mM NaCl, pH 7.0 - CHT Pre-equilibration andElution Buffer Component Molarity (mM) g/L Sodium Phosphate (monobasic)57.5 7.94 Sodium phosphate (dibasic-12 hydrate) 67.5 24.2 SodiumChloride 25 1.46 pH 6.8-7.2 Conductivity (mS/cm at 24-26° C.) 15-20

TABLE 8 10 mM Phosphate 500 mM NaCl, pH 7.0 - CHT Wash 1 BufferComponent Molarity (mM) g/L Sodium Phosphate (monobasic) 3.0 5.82 Sodiumphosphate (dibasic-12 hydrate) 7.0 0.04 Sodium Chloride 500 29.2 pH6.8-7.2 Conductivity (mS/cm at 24-26° C.) 43-53

TABLE 9 10 mM Citrate, pH 3.5 - Low pH VI Hold Buffer Component Molarity(mM) g/L Sodium Citrate Dihydrate 15 1.50 Anhydrous Citric Acid 5.0 2.83pH 3.4-3.6 Conductivity (mS/cm at 24-26° C.) 1.0-3.0

TABLE 10 1× Phosphate Buffered Saline, pH 7.4 - Final TFF BufferComponent Molarity (mM) g/L Disodium Hydrogen Phosphate 10.0 1.44Potassium Dihydrogen Phosphate 2.0 0.24 Sodium Chloride 137 8.00Potassium Chloride 2.7 0.20 pH 7.2-7.6 Conductivity (mS/cm at 24-26° C.)13-17

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

We claim:
 1. An automated method for the-production of cells and cellproducts and purification thereof in a contaminant-free environment,comprising the steps of: providing a first disposable culturewaremodule, the first disposable cultureware module including a cell growthchamber; providing a first reusable instrumentation base deviceincorporating hardware to support cell culture growth, the firstreusable instrumentation base device including a microprocessor controland a pump for circulating cell culture medium through the cell growthchamber; wherein the first disposable cultureware module and the firstreusable instrumentation base device are components of a cell cultureunit; providing a second disposable cultureware module, the seconddisposable cultureware module including a selection device; providing asecond reusable instrumentation base device incorporating hardware toreceive fluid from the cell growth chamber, wherein the seconddisposable cultureware module and the second reusable instrumentationbase device are components of a purification unit; wherein the pumpcomprises dual pumps for chromatography comprising a first pump and asecond pump, and wherein the hardware of the first reusableinstrumentation base device further comprises a plurality of valves,wherein the cell growth chamber includes ports for movement of cellculture medium through the cell growth chamber, wherein the dual pumpsand the plurality of valves circulate cell culture medium into and outof the cell growth chamber through the ports of the cell growth chamber,wherein the cell culture unit and the purification unit are connected bya fluid flow path there between, wherein the hardware of the secondreusable instrumentation device receives fluid from the cell growthchamber through the fluid flow path, wherein the selection devicecomprises a chromatography device, wherein the second disposablecultureware module further includes a diafiltration module that utilizestangential flow filtration, an anion exchange filter, and asize-exclusion virus filter, wherein the chromatography device,diafiltration module, anion exchange filter, and size-exclusion virusfilter are connected to the cell growth chamber of the cell culture unitby the fluid flow path, wherein the chromatography device separates acomponent from fluid received from the cell growth chamber, wherein thediafiltration module separates a component from fluid received from thechromatography device, wherein the anion exchange filter separates acomponent from fluid received from the diafiltration module or thechromatography device, and wherein the size-exclusion virus filterseparates virus from fluid received from the chromatography device, thediafiltration module, or the anion exchange filter, removably attachingthe first disposable cultureware module to the first reusableinstrumentation base device; introducing cells into the cell growthchamber; fluidly attaching a source of cell culture medium to the firstdisposable cultureware module; programming operating parameters into themicroprocessor control; operating the dual pumps to circulate the cellculture medium through the cell growth chamber to grow cells or cellproducts therein; loading the culture medium containing the cellproducts onto the chromatography device to absorb a cell product ontothe chromatography device; eluting the absorbed cell product into anaqueous medium; and collecting the cell product in a pre-sterilizeddisposable collection vessel to form a purified cell product.
 2. Themethod of claim 1, wherein the chromatography device is selected fromthe group consisting of an affinity chromatography device,immuno-affinity chromatography device, ionic exchange chromatographydevice, hydrophobic interaction chromatography device, and sizeselection chromatography (SEC) device, or a combination of two or moreof the foregoing.
 3. The method of claim 1, wherein the chromatographydevice comprises hydroxyapatite resin.
 4. The method of claim 1, whereinthe chromatography device comprises a column packed with an affinityresin.
 5. The method of claim 4, wherein the affinity resin is anti-IgMresin.
 6. The method of claim 4, wherein the affinity resin is selectedfrom the group consisting of a Protein A, Protein G, and anti-IgG resin.7. The method of claim 1, wherein the cell product is an antibody. 8.The method of claim 7, wherein the antibody comprises IgM, IgG, IgD,IgE, or IgA.
 9. The method of claim 7, wherein the antibody comprisesIgM.
 10. The method of claim 7, wherein the antibody targets an idiotypeantigen expressed on B cell tumors.
 11. The method of claim 7, whereinthe antibody targets a tumor antigen.
 12. The method of claim 1, whereinthe cell product is a protein.
 13. The method of claim 1, furthercomprising conjugating the cell product to an adjuvant.
 14. The methodof claim 13, wherein the adjuvant is keyhole limpet hemocyanin, albumin,or β₂-glycoprotein I.